Diagnosis and treatment of aur1 and/or aur2 related disorders

ABSTRACT

The present invention relates to AUR1 and/or AUR2 polypeptides, nucleic acids encoding such polypeptides, cells, tissues and animals containing such nucleic acids, antibodies to such polypeptides, assays utilizing such polypeptides, and methods relating to all of the foregoing. Methods for treatment, diagnosis, and screening are provided for AUR1 and/or AUR2 related diseases or conditions characterized by an abnormal interaction between a AUR1 and/or AUR2 polypeptide and a AUR1 and/or AUR2 binding partner.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. to be assigned (Lyon & Lyon Docket No. 229/022),filed Jan. 9, 1998, by Plowman et al., and entitled “AUR1 and/or AUR2Related Disorders” which is a continuation-in-part of U.S. patentapplication Ser. No. 08/755,728 (Lyon & Lyon Docket No. 223/113), filedNov. 25, 1996, by Plowman, et al., and entitled “Diagnosis and Treatmentof AUR1 and/or AUR2 Related Disorders”, and relates to U.S. patentapplication Ser. No. 60/008,809 filed Dec. 18, 1995, and U.S. patentapplication Ser. No. 60/023,943, filed Aug. 14, 1996, all of which areincorporated herein by reference in their entirety, including anydrawings.

FIELD OF THE INVENTION

[0002] The present invention relates to the novel proteins termed AURORAONE and AURORA TWO (“AUR1 and AUR2”), nucleotide sequences encoding AUR1and/or AUR2, as well as various products and methods useful for thediagnosis and treatment of various AUR1 and/or AUR2 related diseases andconditions.

BACKGROUND OF THE INVENTION

[0003] The following description of the background of the invention isprovided to aid in understanding the invention but is not admitted to beprior art to the invention.

[0004] Cellular signal transduction is a fundamental mechanism wherebyexternal stimuli that regulate diverse cellular processes are relayed tothe interior of cells. One of the key biochemical mechanisms of signaltransduction involves the reversible phosphorylation of proteins, whichenables regulation of the activity of mature proteins by altering theirstructure and function.

[0005] The best characterized protein kinases in eukaryotesphosphorylate proteins on the alcohol moiety of serine, threonine andtyrosine residues. These kinases largely fall into two groups, thosespecific for phosphorylating serines and threonines, and those specificfor phosphorylating tyrosines. Some kinases, referred to as “dualspecificity” kinases, are able to phosphorylate on tyrosine as well asserine/threonine residues.

[0006] Protein kinases can also be characterized by their locationwithin the cell. Some kinases are transmembrane receptor-type proteinscapable of directly altering their catalytic activity in response to theexternal environment such as the binding of a ligand. Others arenon-receptor-type proteins lacking any transmembrane domain. They can befound in a variety of cellular compartments from the inner surface ofthe cell membrane to the nucleus.

[0007] Many kinases are involved in regulatory cascades wherein theirsubstrates may include other kinases whose activities are regulated bytheir phosphorylation state. Ultimately the activity of some downstreameffector is modulated by phosphorylation resulting from activation ofsuch a pathway.

[0008] The serine/threonine kinase family includes members found at allsteps of various signaling cascades, including those involved incontrolling cell growth, migration, differentiation and secretion ofhormones, phosphorylation of transcription factors resulting in alteredgene expression, muscle contraction, glucose metabolism, control ofcellular protein synthesis, and regulation of the cell cycle.

[0009] Chromosomal abnormalities are a hallmark of human cancer,reflecting the deleterious consequences of the gain or loss of geneticinformation (Mitelman et al., Nature Genet. 15:417-474, 1997; Hartwellet al., Science 266:1821-1828, 1994). Some of these defects may have acausal role in cellular transformation due to loss of a negative growthregulator, loss of a gene responsible for maintenance of genomeintegrity, or through the amplification or activation of an oncogene(Kinzler et al., Nature 386:761-763, 1997; Hunter Cell 88:333-346,1997). Alternatively, these abnormalities may be a consequence of tumorprogression where mitotic checkpoints have been disrupted, resulting inabnormal nuclei, miss-segregated chromosomes, and aneuploidy (ElledgeScience 274:1664-1672, 1996; Sherr Science 274:1672-1677, 1996).

SUMMARY OF THE INVENTION

[0010] The present invention relates in part to AUR1 and/or AUR2polypeptides, nucleic acids encoding such polypeptides, cells, tissuesand animals containing such nucleic acids, antibodies to suchpolypeptides, assays utilizing such polypeptides, and methods relatingto all of the foregoing. The utility of the present invention includesthe ability to screen for inhibitors of cell growth and to develop smallmolecule therapeutics for treating cancers.

[0011] Thus, in a first aspect, the invention features an isolated,enriched, or purified nucleic acid encoding an AUR1 and/or AUR2polypeptide.

[0012] By “isolated” in reference to nucleic acid is meant a polymer of6 (preferably 21, more preferably 39, most preferably 75) or morenucleotides conjugated to each other, including DNA and RNA that isisolated from a natural source or that is synthesized. In certainembodiments of the invention, longer nucleic acids are preferred, forexample those of 300, 600, 900 or more nucleotides and/or those havingat least 50%, 60%, 75%, 90%, 95% or 99% identity to the full lengthsequence shown in SEQ ID NO:1 or SEQ ID NO:2. The isolated nucleic acidof the present invention is unique in the sense that it is not found ina pure or separated state in nature. Use of the term “isolated”indicates that a naturally occurring sequence has been removed from itsnormal cellular (i.e., chromosomal) environment. Thus, the sequence maybe in a cell-free solution or placed in a different cellularenvironment. The term does not imply that the sequence is the onlynucleotide chain present, but that it is essentially free (about 90-95%pure at least) of non-nucleotide material naturally associated with it,and thus is distinguished from isolated chromosomes.

[0013] By the use of the term “enriched” in reference to nucleic acid ismeant that the specific DNA or RNA sequence constitutes a significantlyhigher fraction (2-5 fold) of the total DNA or RNA present in the cellsor solution of interest than in normal or diseased cells or in the cellsfrom which the sequence was taken. This could be caused by a person bypreferential reduction in the amount of other DNA or RNA present, or bya preferential increase in the amount of the specific DNA or RNAsequence, or by a combination of the two. However, it should be notedthat enriched does not imply that there are no other DNA or RNAsequences present, just that the relative amount of the sequence ofinterest has been significantly increased. The term significant here isused to indicate that the level of increase is useful to the personmaking such an increase, and generally means an increase relative toother nucleic acids of about at least 2 fold, more preferably at least 5to 10 fold or even more. The term also does not imply that there is noDNA or RNA from other sources. The other source DNA may, for example,comprise DNA from a yeast or bacterial genome, or a cloning vector suchas pUC19. This term distinguishes from naturally occurring events, suchas viral infection, or tumor type growths, in which the level of onemRNA may be naturally increased relative to other species of mRNA. Thatis, the term is meant to cover only those situations in which a personhas intervened to elevate the proportion of the desired nucleic acid.

[0014] It is also advantageous for some purposes that a nucleotidesequence be in purified form. The term “purified” in reference tonucleic acid does not require absolute purity (such as a homogeneouspreparation). Instead, it represents an indication that the sequence isrelatively more pure than in the natural environment (compared to thenatural level this level should be at least 2-5 fold greater, e.g., interms of mg/mL). Individual clones isolated from a cDNA library may bepurified to electrophoretic homogeneity. The claimed DNA moleculesobtained from these clones could be obtained directly from total DNA orfrom total RNA. The cDNA clones are not naturally occurring, but ratherare preferably obtained via manipulation of a partially purifiednaturally occurring substance (messenger RNA). The construction of acDNA library from mRNA involves the creation of a synthetic substance(cDNA) and pure individual cDNA clones can be isolated from thesynthetic library by clonal selection of the cells carrying the cDNAlibrary. Thus, the process which includes the construction of a cDNAlibrary from mRNA and isolation of distinct cDNA clones yields anapproximately 10⁶-fold purification of the native message. Thus,purification of at least one order of magnitude, preferably two or threeorders, and more preferably four or five orders of magnitude isexpressly contemplated.

[0015] By “an AUR1 and/or AUR2 polypeptide” is meant 25 (preferably 30,more preferably 35, most preferably 40) or more contiguous amino acidsset forth in the full length amino acid sequence of SEQ ID NO:3 or SEQID NO:4, or a functional derivative thereof as described herein. Incertain aspects, polypeptides of 100, 200, 300 or more amino acids arepreferred. The AUR1 and/or AUR2 polypeptide can be encoded by afull-length nucleic acid sequence or any portion of the full-lengthnucleic acid sequence, so long as a functional activity of thepolypeptide is retained.

[0016] Also included are inactive and activated mutants of AUR1 and/orAUR2, including, but not limited to those defined in Example 11 herein.By “inactive” is meant an AUR1 and/or AUR2 polypeptide which lackskinase activity. In some embodiments, the essential lysine (residue 162)is mutated. Preferably the polypeptide is otherwise unchanged. By“activated” is meant an AUR1 and/or AUR2 polypeptide which has kinaseactivity in vitro, preferably in situations where the unmutatedpolypeptide does not. Preferably, the AUR1 and/or AUR2 polypeptide ismutated to mimic constitutive phosphorylation. In some embodiments, thethreonine at residue 288 in the activation loop is modified to anaspartic acid.

[0017] The amino acid sequence will be substantially similar to thesequence shown in SEQ ID NO:3 or SEQ ID NO:4, or fragments thereof. Asequence that is substantially similar will preferably have at least 90%identity (more preferably at least 95% and most preferably 99-100%) tothe sequence of SEQ ID NO:3 or SEQ ID NO:4.

[0018] By “identity” is meant a property of sequences that measurestheir similarity or relationship. Identity is measured by dividing thenumber of identical residues by the total number of residues andmultiplying the product by 100. Thus, two copies of exactly the samesequence have 100% identity, but sequences that are less highlyconserved, and have deletions, additions, or replacements, may have alower degree of identity. Those skilled in the art will recognize thatseveral computer programs are available for determining sequenceidentity.

[0019] In a preferred embodiment, the invention features a nucleic acidmolecule comprising a nucleotide sequence that: (a) encodes apolypeptide having the full length amino acid sequence set forth in SEQID NO:3 or SEQ ID NO:4; (b) is the complement of the nucleotide sequenceof (a); (c) hybridizes under highly stringent conditions to the nucleicacid molecule of (a) and encodes a naturally occurring AUR1 and/or AUR2polypeptide; (d) encodes AUR1 and/or AUR2 polypeptide having the fulllength amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4except that it lacks one or more of the following segments of amino acidresidues: 1-73, 74-271, or 272-344 of SEQ ID NO:3, or 1-129, 130-274, or275-403 of SEQ ID NO:4; (e) is the complement of the nucleotide sequenceof (d); (f) encodes a polypeptide having the amino acid sequence setforth in SEQ ID NO:3 or SEQ ID NO:4 from amino acid residues 1-73,74-271, or 272-344 of SEQ ID NO:3, or 1-129, 130-274, 275-403 of SEQ IDNO:4; (g) is the complement of the nucleotide sequence of (f); (h)encodes a polypeptide having the full length amino acid sequence setforth in SEQ ID NO:3 or SEQ ID NO:4 except that it lacks one or more ofthe domains selected from the group consisting of a C-terminal domain, acatalytic domain, and an N-terminal domain; or (i) is the complement ofthe nucleotide sequence of (h).

[0020] The term “complement” refers to two nucleotides that can formmultiple favorable interactions with one another. For example, adenineis complementary to thymine as they can form two hydrogen bonds.Similarly, guanine and cytosine are complementary since they can formthree hydrogen bonds. A nucleotide sequence is the complement of anothernucleotide sequence if all of the nucleotides of the first sequence arecomplementary to all of the nucleotides of the second sequence.

[0021] The term “domain” refers to a region of a polypeptide whichcontains a particular function. For instance, N-terminal or C-terminaldomains of signal transduction proteins can serve functions including,but not limited to, binding molecules that localize the signaltransduction molecule to different regions of the cell or binding othersignaling molecules directly responsible for propagating a particularcellular signal. Some domains can be expressed separately from the restof the protein and function by themselves, while others must remain partof the intact protein to retain function. The latter are termedfunctional regions of proteins and also relate to domains.

[0022] The term “N-terminal domain” refers to a portion of the fulllength amino acid sequence spanning from the amino terminus to the startof the catalytic domain. The N-terminal domain spans amino acid residues1-73 of the sequence set forth in SEQ ID NO:3 or amino acids 1-130 ofthe sequence set forth in SEQ ID NO:4.

[0023] The term “catalytic domain” refers to a portion of the fulllength amino acid sequence that does not contain the N-terminal domainor the C-terminal domain and has catalytic activity. The catalyticdomain spans amino acid residues 73-271 of the sequence set forth in SEQID NO:3 or residues 130-274 of the sequence set forth in SEQ ID NO:4.

[0024] The term “C-terminal domain” refers to a portion of the fulllength amino acid sequence that begins at the end of the catalyticdomain and ends at the carboxyl terminal amino acid, which is the lastamino acid encoded before the stop codon in the nucleic acid sequence.The C-terminal domain spans amino acid residues 272-344 of the sequenceset forth in SEQ ID NO:3 or amino acids 275-403 of the sequence setforth in SEQ ID NO:4.

[0025] In preferred embodiments, the isolated nucleic acid comprises,consists essentially of, or consists of a nucleic acid sequence setforth in SEQ ID NO:1 or SEQ ID NO:2, encodes the full length amino acidsequence of SEQ ID NO:3 or SEQ ID NO:4, a functional derivative thereof,or at least 25, 30, 35, 40, 50, 100, 200, or 300 contiguous amino acidsthereof. The AUR1 and/or AUR2 polypeptide comprises, consistsessentially of, or consists of at least 25, 30, 35, or 40 contiguousamino acids of an AUR1 and/or AUR2 polypeptide. The nucleic acid may beisolated from a natural source by cDNA cloning or by subtractivehybridization. The natural source may be mammalian, preferably human,blood, semen, or tissue and the nucleic acid may be synthesized by thetriester method or by using an automated DNA synthesizer.

[0026] In yet other preferred embodiments, the nucleic acid is aconserved or unique region, for example those useful for: the design ofhybridization probes to facilitate identification and cloning ofadditional polypeptides, the design of PCR probes to facilitate cloningof additional polypeptides, obtaining antibodies to polypeptide regions,and designing antisense oligonucleotides. Examples of amino acidsequences of the present invention include the following amino acidsequences (the isolated, purified or enriched nucleic acids encodingthem are also within the scope of the present invention): ENSYPWPYGRQ(SEQ ID NO:5), CISGP (SEQ ID NO:6), QFPQ (SEQ ID NO:7), VNSGQ (SEQ IDNO:8), RKEPVTPSA-LV (SEQ ID NO:9), LMSRSNVQPTAAP (SEQ ID NO:10),VQNQKQKQLQATSVPH (SEQ ID NO:11), PVSRPLNNTQK (SEQ ID NO:12), VMENSSGTPD(SEQ ID NO:13), ILTRHFTID (SEQ ID NO:14), and SKQPLPSAPENNPEEQLASKQK(SEQ ID NO:15).

[0027] By “conserved nucleic acid regions”, are meant regions present ontwo or more nucleic acids encoding an AUR1 and/or AUR2 polypeptide, towhich a particular nucleic acid sequence can hybridize under lowerstringency conditions. Examples of lower stringency conditions suitablefor screening for nucleic acid encoding AUR1 and/or AUR2 polypeptidesare provided in Abe et al. J. Biol. Chem. 19:13361-13368, 1992 (herebyincorporated by reference herein in its entirety, including anydrawings). Preferably, conserved regions differ by no more than 5 out of20 nucleotides.

[0028] By “unique nucleic acid region” is meant a sequence present in afull length nucleic acid coding for an AUR1 and/or AUR2 polypeptide thatis not present in a sequence coding for any other naturally occurringpolypeptide. Such regions preferably comprise 30 to 45 contiguousnucleotides present in the full length nucleic acid encoding an AUR1and/or AUR2 polypeptide. In particular, a unique nucleic acid region ispreferably of mammalian origin.

[0029] In a preferred embodiment, the isolated, enriched or purifiednucleic acid molecule encoding AUR1 and/or AUR2 polypeptide, comprises avector or promoter effective to initiate transcription in a host cell.

[0030] The invention also features a nucleic acid probe for thedetection of nucleic acid encoding an AUR1 and/or AUR2 polypeptide in asample. The nucleic acid probe contains a nucleotide base sequence thatwill hybridize to a sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 ora functional derivative thereof.

[0031] In preferred embodiments the nucleic acid probe hybridizes tonucleic acid encoding at least 12, 75, 90, 105, 120, 150, 200, 250, 300or 350 contiguous amino acids of the full-length sequence set forth inSEQ ID NO:3 or SEQ ID NO:4 or a functional derivative thereof. Variouslow or high stringency hybridization conditions may be used dependingupon the specificity and selectivity desired. Under stringenthybridization conditions only highly complementary nucleic acidsequences hybridize. Preferably, such conditions prevent hybridizationof nucleic acids having 1 or 2 mismatches out of 20 contiguousnucleotides.

[0032] By stringent hybridization assay conditions is meanthybridization assay conditions at least as stringent as the following:hybridization in 50% formamide, 5×SSC, 50 mM NaH₂PO₄, pH 6.8, 0.5% SDS,0.1 mg/mL sonicated salmon sperm DNA, and 5×Denhart solution at 42° C.overnight; washing with 2×SSC, 0.1% SDS at 45° C.; and washing with0.2×SSC, 0.1% SDS at 45° C.

[0033] Methods for using the probes include detecting the presence oramount of AUR1 and/or AUR2 RNA in a sample by contacting the sample witha nucleic acid probe under conditions such that hybridization occurs anddetecting the presence or amount of the probe bound to AUR1 and/or AUR2RNA. The nucleic acid duplex formed between the probe and a nucleic acidsequence coding for an AUR1 and/or AUR2 polypeptide may be used in theidentification of the sequence of the nucleic acid detected (Nelson etal., in Nonisotopic DNA Probe Techniques, Academic Press, San Diego,Kricka, ed., p. 275, 1992, hereby incorporated by reference herein inits entirety, including any drawings). Kits for performing such methodsmay be constructed to include a container means having disposed thereina nucleic acid probe.

[0034] The invention also features recombinant nucleic acid, preferablyin a cell or an organism. The recombinant nucleic acid may contain asequence set forth in SEQ ID NO:1 or SEQ ID NO:2 or a functionalderivative thereof and a vector or a promoter effective to initiatetranscription in a host cell. The recombinant nucleic acid canalternatively contain a transcriptional initiation region functional ina cell, a sequence complementary to an RNA sequence encoding an AUR1and/or AUR2 polypeptide and a transcriptional termination regionfunctional in a cell.

[0035] In another aspect, the invention describes a recombinant cell ortissue containing nucleic acid coding for an AUR1 and/or AUR2polypeptide. In such cells, the nucleic acid may be under the control ofits genomic regulatory elements, or may be under the control ofexogenous regulatory elements including an exogenous promoter. By“exogenous” it is meant a promoter that is not normally coupled in vivotranscriptionally to the coding sequence for the AUR1 and/or AUR2polypeptide.

[0036] The polypeptide is preferably a fragment of the protein encodedby the full length amino acid sequence set forth in SEQ ID NO:3 or SEQID NO:4. By “fragment,” is meant an amino acid sequence present in afull-length AUR1 and/or AUR2 polypeptide that is not present in anyother naturally occurring polypeptide. Preferably, such a sequencecomprises 6 contiguous amino acids present in the full sequence. Morepreferably, such a sequence comprises 12 contiguous amino acids presentin the full sequence. Even more preferably, such a sequence comprises 18contiguous amino acids present in the full sequence.

[0037] In another aspect the invention features an isolated, enriched,or purified AUR1 and/or AUR2 polypeptide.

[0038] By “isolated” in reference to a polypeptide is meant a polymer of2 (preferably 7, more preferably 13, most preferably 25) or more aminoacids conjugated to each other, including polypeptides that are isolatedfrom a natural source or that are synthesized. In certain aspects longerpolypeptides are preferred, such as those with 402, 407, 413, or 425contiguous amino acids set forth in SEQ ID NO:3 or SEQ ID NO:4. Theisolated polypeptides of the present invention are unique in the sensethat they are not found in a pure or separated state in nature. Use ofthe term “isolated” indicates that a naturally occurring sequence hasbeen removed from its normal cellular environment. Thus, the sequencemay be in a cell-free solution or placed in a different cellularenvironment. The term does not imply that the sequence is the only aminoacid chain present, but that it is essentially free (about 90-95% pureat least) of non-amino acid material naturally associated with it.

[0039] By the use of the term “enriched” in reference to a polypeptideis meant that the specific amino acid sequence constitutes asignificantly higher fraction (2-5 fold) of the total amino acidspresent in the cells or solution of interest than in normal or diseasedcells or in the cells from which the sequence was taken. This could becaused by a person by preferential reduction in the amount of otheramino acids present, or by a preferential increase in the amount of thespecific amino acid sequence of interest, or by a combination of thetwo. However, it should be noted that enriched does not imply that thereare no other amino acid sequences present, just that the relative amountof the sequence of interest has been significantly increased. The termsignificant here is used to indicate that the level of increase isuseful to the person making such an increase, and generally means anincrease relative to other amino acids of about at least 2 fold, morepreferably at least 5 to 10 fold or even more. The term also does notimply that there is no amino acid from other sources. The other sourceamino acid may, for example, comprise amino acid encoded by a yeast orbacterial genome, or a cloning vector such as pUC19. The term is meantto cover only those situations in which man has intervened to elevatethe proportion of the desired amino acid.

[0040] It is also advantageous for some purposes that an amino acidsequence be in purified form. The term “purified” in reference to apolypeptide does not require absolute purity (such as a homogeneouspreparation); instead, it represents an indication that the sequence isrelatively purer than in the natural environment. Compared to thenatural level this level should be at least 2-5 fold greater (e.g., interms of mg/mL). Purification of at least one order of magnitude,preferably two or three orders, and more preferably four or five ordersof magnitude is expressly contemplated. The substance is preferably freeof contamination at a functionally significant level, for example 90%,95%, or 99% pure.

[0041] In preferred embodiments, the AUR1 and/or AUR2 polypeptidecontains at least 25, 30, 35, 40, 50, 100, 150, 200, 250, 300, or 350contiguous amino acids of the full-length sequence set forth in SEQ IDNO:3 or SEQ ID NO:4, or a functional derivative thereof.

[0042] Also included are inactive and activated mutants of AUR1 and/orAUR2, including, but not limited to those defined in Example 11 herein.By “inactive” is meant an AUR1 and/or AUR2 polypeptide which lackskinase activity. In some embodiments, the essential lysine (residue 162)is mutated. Preferably the polypeptide is otherwise unchanged. By“activated” is meant an AUR1 and/or AUR2 polypeptide which has kinaseactivity in vitro, preferably in situations where the unmutatedpolypeptide does not. Preferably, the AUR1 and/or AUR2 polypeptide ismutated to mimic constitutive phosphorylation. In some embodiments, thethreonine at residue 288 in the activation loop is modified to anaspartic acid.

[0043] The polypeptide may be isolated from a natural source by methodswell-known in the art. The natural source may be mammalian, preferablyhuman, blood, semen,. or tissue, and the polypeptide may be synthesizedusing an automated polypeptide synthesizer.

[0044] In a preferred embodiment, the invention features a polypeptidecomprising an amino acid sequence having (a) the full length amino acidsequence set forth in SEQ ID NO:3 or SEQ ID NO:4; (b) the full lengthamino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4 except thatit lacks one or more of the following segments of amino acid residues:1-73, 74-271, or 272-344 of SEQ ID NO:3, or 1-129, 130-274, or 275-403of SEQ ID NO:4; (c) the amino acid sequence set forth in SEQ ID NO:3 orSEQ ID NO:4 from amino acid residues 1-73, 74-271, or 272-344 of SEQ IDNO:3, or 1-129, 130-274, or 275-403 of SEQ ID NO:4; or (d) the fulllength amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4except that it lacks one or more of the domains selected from the groupconsisting of a C-terminal domain, a catalytic domain, and an N-terminaldomain.

[0045] In some embodiments the invention includes a recombinant AUR1and/or AUR2 polypeptide. By “recombinant AUR1 and/or AUR2 polypeptide”is meant a polypeptide produced by recombinant DNA techniques such thatit is distinct from a naturally occurring polypeptide either in itslocation (e.g., present in a different cell or tissue than found innature), purity or structure. Generally, such a recombinant polypeptidewill be present in a cell in an amount different from that normallyobserved in nature.

[0046] In yet another aspect, the invention features an antibody (e.g.,a monoclonal or polyclonal antibody) having specific binding affinity toan AUR1 and/or AUR2 polypeptide or an AUR1 and/or AUR2 polypeptidedomain or fragment. By “specific binding affinity” is meant that theantibody binds to the target (AUR1 and/or AUR2) polypeptide with greateraffinity than it binds to other polypeptides under specified conditions.Antibodies or antibody fragments are polypeptides which contain regionsthat can bind other polypeptides. The term “specific binding affinity”describes an antibody that binds to an AUR1 and/or AUR2 polypeptide withgreater affinity than it binds to other polypeptides under specifiedconditions.

[0047] The term “polyclonal” refers to antibodies that are heterogenouspopulations of antibody molecules derived from the sera of animalsimmunized with an antigen or an antigenic functional derivative thereof.For the production of polyclonal antibodies, various host animals may beimmunized by injection with the antigen. Various adjuvants may be usedto increase the immunological response, depending on the host species.

[0048] “Monoclonal antibodies” are substantially homogenous populationsof antibodies to a particular antigen. They may be obtained by anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. Monoclonal antibodies may be obtainedby methods known to those skilled in the art (Kohler et al., Nature256:495-497, 1975, and U.S. Pat. No. 4,376,110).

[0049] The term “antibody fragment” refers to a portion of an antibody,often the hyper variable region and portions of the surrounding heavyand light chains, that displays specific binding affinity for aparticular molecule. A hyper variable region is a portion of an antibodythat physically binds to the polypeptide target.

[0050] Antibodies or antibody fragments having specific binding affinityto an AUR1 and/or AUR2 polypeptide may be used in methods for detectingthe presence and/or amount of AUR1 and/or AUR2 polypeptide in a sampleby probing the sample with the antibody under conditions suitable forAUR1 and/or AUR2-antibody immunocomplex formation and detecting thepresence and/or amount of the antibody conjugated to the AUR1 and/orAUR2 polypeptide. Diagnostic kits for performing such methods may beconstructed to include antibodies or antibody fragments specific forAUR1 and/or AUR2 as well as a conjugate of a binding partner of theantibodies or the antibodies themselves.

[0051] An antibody or antibody fragment with specific binding affinityto an AUR1 and/or AUR2 polypeptide can be isolated, enriched, orpurified from a prokaryotic or eukaryotic organism. Routine methodsknown to those skilled in the art enable production of antibodies orantibody fragments, in both prokaryotic and eukaryotic organisms.Purification, enrichment, and isolation of antibodies, which arepolypeptide molecules, are described above.

[0052] Antibodies having specific binding affinity to an AUR1 and/orAUR2 polypeptide may be used in methods for detecting the presenceand/or amount of AUR1 and/or AUR2 polypeptide in a sample by contactingthe sample with the antibody under conditions such that an immunocomplexforms and detecting the presence and/or amount of the antibodyconjugated to the AUR1 and/or AUR2 polypeptide. Diagnostic kits forperforming such methods may be constructed to include a first containercontaining the antibody and a second container having a conjugate of abinding partner of the antibody and a label, such as, for example, aradioisotope. The diagnostic kit may also include notification of an FDAapproved use and instructions therefor.

[0053] In another aspect, the invention features a hybridoma whichproduces an antibody having specific binding affinity to an AUR1 and/orAUR2 polypeptide or an AUR1 and/or AUR2 polypeptide domain. By“hybridoma” is meant an immortalized cell line which is capable ofsecreting an antibody, for example an antibody to AUR1 and/or AUR2. Inpreferred embodiments the antibody to AUR1 and/or AUR2 comprises asequence of amino acids that is able to specifically bind an AUR1 and/orAUR2 polypeptide.

[0054] In another aspect, the invention features an AUR1 and/or AUR2polypeptide binding agent able to bind to an AUR1 and/or AUR2polypeptide. The binding agent is preferably a purified antibody whichrecognizes an epitope present on an AUR1 and/or AUR2 polypeptide. Otherbinding agents include molecules which bind to the AUR1 and/or AUR2polypeptide and analogous molecules which bind to an AUR1 and/or AUR2polypeptide. Such binding agents may be identified by using assays thatmeasure AUR1 and/or AUR2 binding partner activity, such as those thatmeasure PDGFR activity.

[0055] The invention features a method for screening for human cellscontaining an AUR1 and/or AUR2 polypeptide or an equivalent sequence.The method involves identifying the novel polypeptide in human cellsusing techniques that are routine and standard in the art, such as thosedescribed herein for identifying AUR1 and/or AUR2 (e.g., cloning,Southern or Northern blot analysis, in situ hybridization, PCRamplification, etc.).

[0056] In another aspect, the invention provides a method foridentifying a substance capable of modulating AUR1 and/or AUR2 activitycomprising the steps of (a) contacting AUR1 and/or AUR2 polypeptide witha test substance; (b) measuring the activity of said polypeptide; and(c) determining whether said substance modulates the activity of saidpolypeptide.

[0057] The term “modulates” refers to the ability of a compound to alterthe function of AUR1 and/or AUR2. A modulator preferably activates orinhibits the activity of AUR1 and/or AUR2 depending on the concentrationof the compound exposed to AUR1 and/or AUR2.

[0058] The term “activates” refers to increasing the cellular activityof AUR1 and/or AUR2. The term “inhibit” refers to decreasing thecellular activity of AUR1 and/or AUR2. AUR1 and/or AUR2 activity ispreferably the interaction with a natural binding partner.

[0059] The term “modulates” also refers to altering the function of AUR1and/or AUR2 by increasing or decreasing the probability that a complexforms between AUR1 and/or AUR2 and a natural binding partner. Amodulator preferably increases the probability that such a complex formsbetween AUR1 and/or AUR2 and the natural binding partner, morepreferably increases or decreases the probability that a complex formsbetween AUR1 and/or AUR2 and the natural binding partner depending onthe concentration of the compound exposed to AUR1 and/or AUR2, and mostpreferably decreases the probability that a complex forms between AUR1and/or AUR2 and the natural binding partner.

[0060] The term “complex” refers to an assembly of at least twomolecules bound to one another. Signal transduction complexes oftencontain at least two protein molecules bound to one another. Forinstance, a protein tyrosine receptor protein kinase, GRB2, SOS, RAF,and RAS assemble to form a signal transduction complex in response to amitogenic ligand.

[0061] The term “natural binding partner” refers to polypeptides ornucleic acids that bind to AUR1 and/or AUR2 in cells. A change in theinteraction between AUR1 and/or AUR2 and a natural binding partner canmanifest itself as an increased or decreased probability that theinteraction forms, or an increased or decreased concentration of AUR1and/or AUR2/natural binding partner complex.

[0062] The term “contacting” as used herein refers to mixing a solutioncomprising the test compound with a liquid medium bathing the cells ofthe methods. The solution comprising the compound may also compriseanother component, such as dimethyl sulfoxide (DMSO), which facilitatesthe uptake of the test compound or compounds into the cells of themethods. The solution comprising the test compound may be added to themedium bathing the cells by utilizing a delivery apparatus, such as apipet-based device or syringe-based device.

[0063] In another aspect, the invention provides for the treatment ofdiseases by administering to a patient in need of such treatment asubstance that modulates the activity of AUR1 and/or AUR2. Suchsubstances preferably show positive results in one or more in vitroassays for an activity corresponding to treatment of the disease ordisorder in question (such as the assays described in example 13 below).Examples of substances that can be screened for favorable activity areprovided in section XI below. The diseases that could be treated by amodulator of AUR1 and/or AUR2 activity preferably include colon, breast,renal, ovarian, bladder, head and neck cancers, and gliomas,medulloblastomas, chondrosarcomas, and pancreatic tumors, and preferablyinclude breast, colon, and renal cancers, and more preferably, coloncancer. The substances that modulate the activity of AUR1 and/or AUR2preferably include, but are not limited to, antisense oligonucleotides,as described herein, and inhibitors of protein kinases, as determined bymethods and screens described herein in the Examples.

[0064] Another aspect of the invention features a method for detectionof aur1 and/or aur2 in a sample as a diagnostic tool for diseasescomprising the steps of (a) contacting said sample with a nucleic acidprobe which hybridizes under hybridization assay conditions to a nucleicacid target region of aur1 and/or aur2, said probe comprising thenucleic acid sequence encoding an AUR1 and/or AUR2 polypeptide, afragment thereof, or the complement of said sequence or fragment; and(b) detecting the presence or amount of the probe:target region hybridas an indication of said disease.

[0065] The aur1 and/or aur2 “target region” is the nucleotide basesequence set forth in SEQ ID NO:1 or SEQ ID NO:2, a functionalderivative thereof, or a fragment thereof to which the nucleic acidprobe will specifically hybridize. Specific hybridization indicates thatin the presence of other nucleic acids the probe only hybridizesdetectably with the aur1 and/or aur2 target region. Putative targetregions can be identified by methods well known in the art consisting ofalignment and comparison of the most closely related sequences in thedatabase.

[0066] In preferred embodiments the nucleic acid probe hybridizes to anaur1 and/or aur2 target region encoding at least 12, 75, 90, 105, 120,150, 200, 250, 300 or 350 contiguous amino acids of the full-lengthsequence set forth in SEQ ID NO:3 or SEQ ID NO:4 or a functionalderivative thereof. Hybridization conditions should be such thathybridization occurs only with aur1 and/or aur2 in the presence of othernucleic acid molecules. Under stringent hybridization conditions onlyhighly complementary nucleic acid sequences hybridize. Preferably, suchconditions prevent hybridization of nucleic acids having 1 or 2mismatches out of 20 contiguous nucleotides. Such conditions are definedsupra.

[0067] The diseases for which detection of aur1 and/or aur2 in a samplecould be diagnostic include diseases in which aur1 and/or aur2 nucleicacid (DNA and/or RNA) is amplified in comparison to normal cells. By“amplification” is meant increased numbers of aur1 and/or aur2 DNA orRNA in a cell compared with normal cells. In normal cells, aur1 and aur2are found as single copy genes. In selected diseases, the chromosomallocation of aur1 and/or aur2 is amplified, resulting in multiple copiesof the gene, or amplification. Gene amplification can lead toamplification of aur1 and/or aur2 RNA, or aur1 and/or aur2 RNA can beamplified in the absence of aur1 and/or aur2 DNA amplification.

[0068] “Amplification” as it refers to RNA can be the detectablepresence of aur1 and/or aur2 RNA in cells, since in some normal cellsthere is no basal expression of aur1 and/or aur2 RNA. In other normalcells, a basal level of expression of aur1 and/or aur2 exists, thereforein these cases amplification is the detection of at least 1-2-fold, andpreferably more, aur1 and/or aur2 RNA, compared to the basal level.

[0069] The diseases that could be diagnosed by detection of aur1 and/oraur2 in a sample preferably include colon, breast, renal, ovarian,bladder, head and neck cancers, and gliomas, medulloblastomas,chondrosarcomas, and pancreatic tumors, and preferably include breast,colon, and renal cancers, and more preferably, colon cancer.

[0070] The test samples suitable for nucleic acid probing methods of thepresent invention include, for example, cells or nucleic acid extractsof cells, or biological fluids. The samples used in the above-describedmethods will vary based on the assay format, the detection method andthe nature of the tissues, cells or extracts to be assayed. Methods forpreparing nucleic acid extracts of cells are well known in the art andcan be readily adapted in order to obtain a sample which is compatiblewith the method utilized.

[0071] Another aspect of the invention features antisenseoligonucleotides to the nucleic acid sequences encoding AUR1 and/or AUR2polypeptides contained in SEQ ID NO:1 and/or SEQ ID NO:2, and fragmentsthereof. In a preferred invention the antisense oligonucleotides aresynthesized as phosphorothionates. In a preferred embodiment theantisense oligonucleotides comprise the following sequences 5′-3′:nucleotides 1743-1763 of aur2: CAGGGCAGAGTGGTCACTTTC (SEQ ID NO:30),nucleotides 42-62 of aur2: CGTCCGCCACTCCGACCAGCC (SEQ ID NO:31),nucleotides 1654-1674 of aur2: TGCAGTCGAACCTTGCCTCCA (SEQ ID NO:32).

[0072] The antisense oligonucleotides of the invention are preferablyused to inhibit AUR1 and/or AUR2 protein expression in vivo in normaland tumor cells. Antisense oligonucleotides can be used either singly orin combination. In a preferred embodiment, either SEQ ID NO:30 and SEQID NO:32 or SEQ ID NO:31 and SEQ ID NO:32 are used jointly. In apreferred embodiment, expression of AUR2 is significantly reduced, andmore preferably reduced to below the limit of detection. In otherpreferred embodiments, treatment with SEQ ID NO:31 and SEQ ID NO:32inhibits growth and/or induces apoptosis in cells. Antisenseoligonucleotides can also be used to inhibit AUR1 and/or AUR2 proteinexpression in human tumor cell xenografts in nude mice. Antisenseoligonucleotides may preferentially be used as a treatment in varioushuman tumors over expressing AUR2.

[0073] Additional antisense oligonucleotides and effective combinationscan be identified by methods well known in the art. Briefly, cells ortissues over expressing aur1 and/or aur2 can be contacted with antisenseoligonucleotides, either singly or in combination, and the expression ofaur1 and/or aur2 RNA, and/or AUR1 and/or AUR2 polypeptide can bedetermined by methods described herein. Preferably, treatment with aur1and/or aur2 causes a decrease in the expression of aur1 and/or aur2 RNAand/or AUR1 and/or AUR2 polypeptide, more preferably expression isdecreased significantly (1 to 2-fold), most preferably expression isdecreased to an undetectable level.

[0074] The summary of the invention described above is non-limiting andother features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments, and from the claims.

DESCRIPTION OF FIGURES

[0075]FIG. 1 shows the sequences for human aur1 and aur2 deduced fromfull length cDNA clones isolated from normal duodenum, pancreaticcarcinoma, and primary colorectal carcinoma libraries. Xenopus p46B(PIR:S53343), Drosophila aurora (PIR:A56220) and S. cerevesiae IPL1(SWISS-PROT:P38991) are included in the alignment. The alignment wasgenerated by also including the two murine (DDBJ:D21099 and GB:U80932),an additional xenopus (PIR:S53342), and two C. elegans (GB:U53336 andGB:U97196) sequences as input into msa, a parallel coded multiplesequence alignment program that was run on MasPar MP2216 supercomputer.Boxed residues are common to three or more of the sequences, shadedresidues represent regions of amino acid similarity between two or moresequences, overlines correspond to the conserved Aurora Box1 and AuroraBox2 sequences, the arrow denotes the start of the C-terminalserine/threonine kinase domain, the circled residue indicates thelocation of a single nucleotide polymorphism described in the text,solid circles correspond to the location of various yeast and Drosophilamutants, and stars denote the site of the kinase inactivating andactivating point mutants described in the text.

DETAILED DESCRIPTION OF THE INVENTION

[0076] The present invention relates in part to AUR1 and/or AUR2polypeptides, nucleic acids encoding such polypeptides, cells containingsuch nucleic acids, antibodies to such polypeptides, assays utilizingsuch polypeptides, and methods relating to all of the foregoing. Thepresent invention is based upon the isolation and characterization ofnew proteins which we have designated AUR1 and/or AUR2. The polypeptidesand nucleic acids may be produced using well known and standardsynthesis techniques when given the sequences presented herein.

[0077] I. Nucleic Acid Encoding AUR1 and/or AUR2 Polypeptides

[0078] Included within the scope of this invention are the functionalequivalents of the herein-described isolated nucleic acid molecules. Thedegeneracy of the genetic code permits substitution of certain codons byother codons which specify the same amino acid and hence would give riseto the same protein. The nucleic acid sequence can vary substantiallysince, with the exception of methionine and tryptophan, the known aminoacids can be coded for by more than one codon. Thus, portions or all ofthe AUR1 and/or AUR2 gene could be synthesized to give a nucleic acidsequence significantly different from that shown in SEQ ID NO:1 or SEQID NO:2. The encoded amino acid sequence thereof would, however, bepreserved.

[0079] In addition, the nucleic acid sequence may comprise a nucleotidesequence which results from the addition, deletion or substitution of atleast one nucleotide to the 5′-end and/or the 3′-end of the nucleic acidformula shown in SEQ ID NO:1 or SEQ ID NO:2 or a derivative thereof. Anynucleotide or polynucleotide may be used in this regard, provided thatits addition, deletion or substitution does not alter the amino acidsequence of SEQ ID NO:3 or SEQ ID NO:4 which is encoded by thenucleotide sequence. For example, the present invention is intended toinclude any nucleic acid sequence resulting from the addition of ATG asan initiation codon at the 5′-end of the inventive nucleic acid sequenceor its derivative, or from the addition of TTA, TAG or TGA as atermination codon at the 3′-end of the inventive nucleotide sequence orits derivative. Moreover, the nucleic acid molecule of the presentinvention may, as necessary, have restriction endonuclease recognitionsites added to its 5′-end and/or 3′-end.

[0080] Such functional alterations of a given nucleic acid sequenceafford an opportunity to promote secretion and/or processing ofheterologous proteins encoded by foreign nucleic acid sequences fusedthereto. All variations of the nucleotide sequence of the AUR1 and/orAUR2 genes and fragments thereof permitted by the genetic code are,therefore, included in this invention.

[0081] Further, it is possible to delete codons or to substitute one ormore codons with codons other than degenerate codons to produce astructurally modified polypeptide, but one which has substantially thesame utility or activity as the polypeptide produced by the unmodifiednucleic acid molecule. As recognized in the art, the two polypeptidesare functionally equivalent, as are the two nucleic acid molecules whichgive rise to their production, even though the differences between thenucleic acid molecules are not related to the degeneracy of the geneticcode.

[0082] II. A Nucleic Acid Probe for the Detection of AUR1 and/or AUR2

[0083] Southern analysis with probes derived from the unique N-terminalregions of aur1 and aur2 indicate that they exist as single copy genesin human cells. However, under low stringency conditions, 1.3 kb and 3.2kb SacI fragments which weakly hybridize to the aur1 probe weredetected.

[0084] A nucleic acid probe of the present invention may be used toprobe an appropriate chromosomal or cDNA library by usual hybridizationmethods to obtain another nucleic acid molecule of the presentinvention. A chromosomal DNA or cDNA library may be prepared fromappropriate cells according to recognized methods in the art (cf.“Molecular Cloning: A Laboratory Manual”, second edition, Cold SpringHarbor Laboratory, Sambrook, Fritsch, & Maniatis, eds., 1989).

[0085] In the alternative, chemical synthesis is carried out in order toobtain nucleic acid probes having nucleotide sequences which correspondto N-terminal and C-terminal portions of the amino acid sequence of thepolypeptide of interest. The synthesized nucleic acid probes may be usedas primers in a polymerase chain reaction (PCR) carried out inaccordance with recognized PCR techniques, essentially according to PCRProtocols, “A Guide to Methods and Applications”, Academic Press,Michael et al., eds., 1990, utilizing the appropriate chromosomal orcDNA library to obtain the fragment of the present invention.

[0086] One skilled in the art can readily design such probes based onthe sequence disclosed herein using methods of computer alignment andsequence analysis known in the art (“Molecular Cloning: A LaboratoryManual”, 1989, supra). The hybridization probes of the present inventioncan be labeled by standard labeling techniques such as with aradiolabel, enzyme label, fluorescent label, biotin-avidin label,chemiluminescence, and the like. After hybridization, the probes may bevisualized using known methods.

[0087] The nucleic acid probes of the present invention include RNA, aswell as DNA probes, such probes being generated using techniques knownin the art. The nucleic acid probe may be immobilized on a solidsupport. Examples of such solid supports include, but are not limitedto, plastics such as polycarbonate, complex carbohydrates such asagarose and sepharose, and acrylic resins, such as polyacrylamide andlatex beads. Techniques for coupling nucleic acid probes to such solidsupports are well known in the art.

[0088] The test samples suitable for nucleic acid probing methods of thepresent invention include, for example, cells or nucleic acid extractsof cells, or biological fluids. The samples used in the above-describedmethods will vary based on the assay format, the detection method andthe nature of the tissues, cells or extracts to be assayed. Methods forpreparing nucleic acid extracts of cells are well known in the art andcan be readily adapted in order to obtain a sample which is compatiblewith the method utilized.

[0089] III. A Probe Based Method And Kit For Detecting AUR1 and/or AUR2

[0090] Aur1 RNA is broadly expressed in rapidly dividing cells derivedfrom both normal and tumor tissues. Aur2 RNA is expressed in a morerestricted pattern, being low or absent in most normal tissues, andabundant in only a subset of tumor-derived cell lines, particularlythose of colon, renal, melanoma, and breast origin in which the 2.4 kbaur2 transcript is expressed in 96% (24 of 25). The 1.4 kb aur1transcript was co-expressed at similar levels as aur2 in the same 24tumor cell lines.

[0091] Aur2 RNA expression is also increased in approximately 54%(22/41) of 41 primary human colorectal tumors compared with matchednormal colorectal controls. Aur2 RNA showed 4-28 fold overexpression intumor versus normal tissue.

[0092] Human aur1 is located on chromosome 17p13.1 and human aur2 onchromosome 20q13.2. Aur2 maps adjacent to the vitamin D hydroxylase(CYP24) gene and the cosmid probe RMC20C001 that lie at 0.825-0.83Flpter (fractional length from pter) on chromosome 20 (Tanner et al.,Cancer res. 54:4257-4260, 1994; Tanner et al., Cancer Res. 56:3441-3445,1996). Both of these markers have been characterized for their presencein the 20q13 amplicon common to many human malignancies, particularlythose from breast, bladder, and colon (Tanner 1994, supra; Tanner 1996,supra; Kallioniemi et al., Proc. Natl. Acad. Sci. USA 91:2156-2160,1994; Yaseen et al., Cancer Genet. Cytogenet. 44:83-97, 1990; Muleris etal., Cancer Genet. Cytogenet. 29:289-301, 1987; Schlegel et al., CancerRes. 55:6002-6005, 1995; James et al., Oncogene 14:1059-1065, 1997;Solinas-Toldo et al., Cancer Res. 56:3803-3807, 1996; Bockmuhl et al.,Laryngorhinootologie 75:408-414, 1996; Larramendy et al., Am. J. Pathol.150:685-691, 1997; Reznikoff et al., Semin. Oncol. 23:571-S84, 1996;Courjal et al., Br. J. Cancer 74:1984-1989, 1996; Iwabuchi et al.,Cancer Res. 55:6172-6180, 1995; Bigner et al., Cancer Genet. Cytogenet.30:91-101, 1988). The aur2-specific bands showed amplification in thetumor samples.

[0093] AUR2 DNA was amplified in 41 of 79 (52%) of the primarycolorectal tumors for which suitable DNA was available for genotyping.Nine of twelve samples demonstrated a 2-8 fold amplification of AUR2 DNAin the tumors compared to normal tissue. Eleven of the samples showed adirect correlation between DNA amplification and RNA overexpression.

[0094] The most common regions of high copy amplification in humanbreast cancer have been localized to 17q22 and 20q13.2 (Tanner 1994,supra; Tanner 1996, supra; Kallioniemi 1994, supra). Low levelamplification of 20q has been described in 6-18% of primary breastcancer and 40% of breast cancer cell lines. The incidence increases to60% in BRCA2 positive breast cancers (Tanner 1994, supra; Tanner 1996,supra; Kallioniemi 1994, supra; Tirkkonen et al., Cancer Res.57:1222-1227, 1997). High levels of 20q amplification correlate withpoor prognosis for patients with node negative breast cancer (Isola etal., Am. J. Pathol. 147:905-911, 1995). Low level amplification of 20qhas also been noted in colon cancer, ovarian cancer, bladder cancer,gliomas, medulloblastomas, chondrosarcomas, pancreatic tumors, and headand neck cancers (Yaseen 1990, supra; Muleris 1987, supra; Schlegel1995, supra; James 1997, supra; Solinas-Toldo 1996, supra; Bockmuhl1996, supra; Larramendy 1997, supra; Reznikoff 1996, supra; Courjal1996, supra; Iwabuchi 1995, supra; Bigner 1988, supra). Several studieshave also found chromosomal gains of 20q in approximately 60% of primarycolorectal carcinomas (Yaseen 1990, supra; Muleris 1987, supra; Schlegel1995, supra). Cell culture models have suggested that low-levelamplification of 20q is associated with immortalization and subsequenthigh-level amplification correlates with chromosomal instability(Savalieva et al., Oncogene 14:551-560, 1997).

[0095] AUR2 DNA was amplified in 41 of 79 (52%) of primary colorectaltumors. The CYP24 gene was coamplified with aur2 in 37 of 41 (90%)matched pairs, and was only once found amplified in the absence of aur2amplification. Aur2 DNA amplification and RNA overexpression is highlycorrelated (r=0.695). DNA amplification may be a mechanism for aur2activation and aur2 may be the oncogene at 20q13 whose high levelamplification correlates with poor clinical outcome in a variety ofsolid tumors (Isola 1995, supra).

[0096] One method of detecting the presence of aur1 and/or aur2 in asample comprises (a) contacting said sample with the above-describednucleic acid probe under conditions such that hybridization occurs, and(b) detecting the presence of said probe bound to said nucleic acidmolecule. One skilled in the art would select the nucleic acid probeaccording to techniques known in the art as described above. Samples tobe tested include but should not be limited to RNA samples of humantissue.

[0097] A kit for detecting the presence of aur1 and/or aur2 in a samplecomprises at least one container means having disposed therein theabove-described nucleic acid probe. The kit may further comprise othercontainers comprising one or more of the following: wash reagents andreagents capable of detecting the presence of bound nucleic acid probe.Examples of detection reagents include, but are not limited toradiolabelled probes, enzymatic labeled probes (horseradish peroxidase,alkaline phosphatase), and affinity labeled probes (biotin, avidin, orsteptavidin).

[0098] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers or strips of plastic orpaper. Such containers allow the efficient transfer of reagents from onecompartment to another compartment such that the samples and reagentsare not cross-contaminated and the agents or solutions of each containercan be added in a quantitative fashion from one compartment to another.Such containers will include a container which will accept the testsample, a container which contains the probe or primers used in theassay, containers which contain wash reagents (such as phosphatebuffered saline, Tris-buffers, and the like), and containers whichcontain the reagents used to detect the hybridized probe, boundantibody, amplified product, or the like. One skilled in the art willreadily recognize that the nucleic acid probes described in the presentinvention can readily be incorporated into one of the established kitformats which are well known in the art.

[0099] IV. DNA Constructs Comprising an Aur1 and/or Aur2 Nucleic AcidMolecule and Cells Containing These Constructs

[0100] The present invention also relates to a recombinant DNA moleculecomprising, 5′ to 3′, a promoter effective to initiate transcription ina host cell and the above-described nucleic acid molecules. In addition,the present invention relates to a recombinant DNA molecule comprising avector and an above-described nucleic acid molecule. The presentinvention also relates to a nucleic acid molecule comprising atranscriptional region functional in a cell, a sequence complementary toan RNA sequence encoding an amino acid sequence corresponding to theabove-described polypeptide, and a transcriptional termination regionfunctional in said cell. The above-described molecules may be isolatedand/or purified DNA molecules.

[0101] The present invention also relates to a cell or organism thatcontains an above-described nucleic acid molecule and thereby is capableof expressing a peptide. The polypeptide may be purified from cellswhich have been altered to express the polypeptide. A cell is said to be“altered to express a desired polypeptide” when the cell, throughgenetic manipulation, is made to produce a protein which it normallydoes not produce or which the cell normally produces at lower levels.One skilled in the art can readily adapt procedures for introducing andexpressing either genomic, cDNA, or synthetic sequences into eithereukaryotic or prokaryotic cells.

[0102] A nucleic acid molecule, such as DNA, is said to be “capable ofexpressing” a polypeptide if it contains nucleotide sequences whichcontain transcriptional and translational regulatory information andsuch sequences are “operably linked” to nucleotide sequences whichencode the polypeptide. An operable linkage is a linkage in which theregulatory DNA sequences and the DNA sequence sought to be expressed areconnected in such a way as to permit gene sequence expression. Theprecise nature of the regulatory regions needed for gene sequenceexpression may vary from organism to organism, but shall in generalinclude a promoter region which, in prokaryotes, contains both thepromoter (which directs the initiation of RNA transcription) as well asthe DNA sequences which, when transcribed into RNA, will signalsynthesis initiation. Such regions will normally include those5′-non-coding sequences involved with initiation of transcription andtranslation, such as the TATA box, capping sequence, CAAT sequence, andthe like.

[0103] If desired, the non-coding region 3′ to the sequence encoding anAUR1 and/or AUR2 polypeptide may be obtained by the above-describedmethods. This region may be retained for its transcriptional terminationregulatory sequences, such as termination and polyadenylation. Thus, byretaining the 3′-region naturally contiguous to the DNA sequenceencoding an AUR1 and/or AUR2 polypeptide, the transcriptionaltermination signals may be provided. Where the transcriptionaltermination signals are not satisfactorily functional in the expressionhost cell, then a 3′ region functional in the host cell may besubstituted.

[0104] Two DNA sequences (such as a promoter region sequence and an aur1and/or aur2 sequence) are said to be operably linked if the nature ofthe linkage between the two DNA sequences does not (1) result in theintroduction of a frame-shift mutation, (2) interfere with the abilityof the promoter region sequence to direct the transcription of an aur1and/or aur2 gene sequence, or (3) interfere with the ability of the aur1and/or aur2 gene sequence to be transcribed by the promoter regionsequence. Thus, a promoter region would be operably linked to a DNAsequence if the promoter were capable of effecting transcription of thatDNA sequence. Thus, to express an aur1 and/or aur2 gene, transcriptionaland translational signals recognized by an appropriate host arenecessary.

[0105] The present invention encompasses the expression of the aur1and/or aur2 gene (or a functional derivative thereof) in eitherprokaryotic or eukaryotic cells. Prokaryotic hosts are, generally, veryefficient and convenient for the production of recombinant proteins andare, therefore, one type of preferred expression system for the aur1and/or aur2 gene. Prokaryotes most frequently are represented by variousstrains of E. coli. However, other microbial strains may also be used,including other bacterial strains.

[0106] In prokaryotic systems, plasmid vectors that contain replicationsites and control sequences derived from a species compatible with thehost may be used. Examples of suitable plasmid vectors may includepBR322, pUC118, pUC119 and the like; suitable phage or bacteriophagevectors may include γgt10, γgt11 and the like; and suitable virusvectors may include pMAM-neo, pKRC and the like. Preferably, theselected vector of the present invention has the capacity to replicatein the selected host cell.

[0107] Recognized prokaryotic hosts include bacteria such as E. coil,Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like.However, under such conditions, the peptide will not be glycosylated.The prokaryotic host must be compatible with the replicon and controlsequences in the expression plasmid.

[0108] To express aur1 and/or aur2 (or a functional derivative thereof)in a prokaryotic cell, it is necessary to operably link the aur1 and/oraur2 sequence to a functional prokaryotic promoter. Such promoters maybe either constitutive or, more preferably, regulatable (i.e., inducibleor derepressible). Examples of constitutive promoters include the intpromoter of bacteriophage λ, the bla promoter of the β-lactamase genesequence of pBR322, and the CAT promoter of the chloramphenicol acetyltransferase gene sequence of pPR325, and the like. Examples of inducibleprokaryotic promoters include the major right and left promoters ofbacteriophage λ (P_(L) and P_(R)), the trp, recA, λacZ, λacI, and galpromoters of E. coli, the α-amylase (Ulmanen et al., J. Bacteriol.162:176-182, 1985) and the ç-28-specific promoters of B. subtilis(Gilman et al., Gene Sequence 32:11-20, 1984), the promoters of thebacteriophages of Bacillus (Gryczan, In: The Molecular Biology of theBacilli, Academic Press, Inc., NY, 1982), and Streptomyces promoters(Ward et al., Mol. Gen. Genet. 203:468-478, 1986). Prokaryotic promotersare reviewed by Glick (Ind. Microbiot. 1:277-282, 1987), Cenatiempo(Biochimie 68:505-516, 1986), and Gottesman (Ann. Rev. Genet.18:415-442, 1984).

[0109] Proper expression in a prokaryotic cell also requires thepresence of a ribosome binding site upstream of the genesequence-encoding sequence. Such ribosome binding sites are disclosed,for example, by Gold et al. (Ann. Rev. Microbial. 35:365-404, 1981). Theselection of control sequences, expression vectors, transformationmethods, and the like, are dependent on the type of host cell used toexpress the gene. As used herein, “cell”, “cell line”, and “cellculture” may be used interchangeably and all such designations includeprogeny. Thus, the words “transformants” or “transformed cells” includethe primary subject cell and cultures derived therefrom, without regardto the number of transfers. It is also understood that all progeny maynot be precisely identical in DNA content, due to deliberate orinadvertent mutations. However, as defined, mutant progeny have the samefunctionality as that of the originally transformed cell.

[0110] Host cells which may be used in the expression systems of thepresent invention are not strictly limited, provided that they aresuitable for use in the expression of the AUR1 and/or AUR2 peptide ofinterest. Suitable hosts may often include eukaryotic cells. Preferredeukaryotic hosts include, for example, yeast, fungi, insect cells,mammalian cells either in vivo, or in tissue culture. Mammalian cellswhich may be useful as hosts include HeLa cells, cells of fibroblastorigin such as VERO or CHO-K1, or cells of lymphoid origin and theirderivatives. Preferred mammalian host cells include SP2/0 and J558L, aswell as neuroblastoma cell lines such as IMR 332, which may providebetter capacities for correct post-translational processing.

[0111] In addition, plant cells are also available as hosts, and controlsequences compatible with plant cells are available, such as thecauliflower mosaic virus 35S and 19S, and nopaline synthase promoter andpolyadenylation signal sequences. Another preferred host is an insectcell, for example the Drosophila larvae. Using insect cells as hosts,the Drosophila alcohol dehydrogenase promoter can be used (Rubin,Science 240:1453-1459, 1988). Alternatively, baculovirus vectors can beengineered to express large amounts of AUR1 and/or AUR2 in insects cells(Jasny, Science 238:1653, 1987; Miller et al., In: Genetic Engineering,Vol. 8, Plenum, Setlow et al., eds., pp. 277-297, 1986).

[0112] Any of a series of yeast expression systems can be utilized whichincorporate promoter and termination elements from the activelyexpressed sequences coding for glycolytic enzymes that are produced inlarge quantities when yeast are grown in mediums rich in glucose. Knownglycolytic gene sequences can also provide very efficienttranscriptional control signals. Yeast provides substantial advantagesin that it can also carry out post-translational modifications. A numberof recombinant DNA strategies exist utilizing strong promoter sequencesand high copy number plasmids which can be utilized for production ofthe desired proteins in yeast. Yeast recognizes leader sequences oncloned mammalian genes and secretes peptides bearing leader sequences(i.e., pre-peptides). Several possible vector systems are available forthe expression of aur1 and/or aur2 in a mammalian host.

[0113] A wide variety of transcriptional and translational regulatorysequences may be employed, depending upon the nature of the host. Thetranscriptional and translational regulatory signals may be derived fromviral sources, such as adenovirus, bovine papilloma virus,cytomegalovirus, simian virus, or the like, where the regulatory signalsare associated with a particular gene sequence which has a high level ofexpression. Alternatively, promoters from mammalian expression products,such as actin, collagen, myosin, and the like, may be employed.Transcriptional initiation regulatory signals may be selected whichallow for repression or activation, so that expression of the genesequences can be modulated. Of interest are regulatory signals which aretemperature-sensitive so that by varying the temperature, expression canbe repressed or initiated, or are subject to chemical (such asmetabolite) regulation.

[0114] Expression of aur1 and/or aur2 in eukaryotic hosts requires theuse of eukaryotic regulatory regions. Such regions will, in general,include a promoter region sufficient to direct the initiation of RNAsynthesis. Preferred eukaryotic promoters include, for example, thepromoter of the mouse metallothionein I gene sequence (Hamer et al., J.Mol. Appl. Gen. 1:273-288, 1982); the TK promoter of Herpes virus(McKnight, Cell 31:355-365, 1982); the SV40 early promoter (Benoist etal., Nature (London) 290:304-31, 1981); and the yeast gal4 gene sequencepromoter (Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975,1982; Silver et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955, 1984).

[0115] Translation of eukaryotic mRNA is initiated at the codon whichencodes the first methionine. For this reason, it is preferable toensure that the linkage between a eukaryotic promoter and a DNA sequencewhich encodes AUR1 and/or AUR2 (or a functional derivative thereof) doesnot contain any intervening codons which are capable of encoding amethionine (i.e., AUG). The presence of such codons results either inthe formation of a fusion protein (if the AUG codon is in the samereading frame as the aur1 and/or aur2 coding sequence) or a frame-shiftmutation (if the AUG codon is not in the same reading frame as the aur1and/or aur2 coding sequence).

[0116] An aur1 and/or aur2 nucleic acid molecule and an operably linkedpromoter may be introduced into a recipient prokaryotic or eukaryoticcell either as a nonreplicating DNA or RNA molecule, which may either bea linear molecule or, more preferably, a closed covalent circularmolecule. Since such molecules are incapable of autonomous replication,the expression of the gene may occur through the transient expression ofthe introduced sequence. Alternatively, permanent expression may occurthrough the integration of the introduced DNA sequence into the hostchromosome.

[0117] A vector may be employed which is capable of integrating thedesired gene sequences into the host cell chromosome. Cells which havestably integrated the introduced DNA into their chromosomes can beselected by also introducing one or more markers which allow forselection of host cells which contain the expression vector. The markermay provide for prototrophy to an auxotrophic host, biocide resistance,e.g., antibiotics, or heavy metals, such as copper, or the like. Theselectable marker gene sequence can either be directly linked to the DNAgene sequences to be expressed, or introduced into the same cell byco-transfection. Additional elements may also be needed for optimalsynthesis mRNA. These elements may include splice signals, as well astranscription promoters, enhancers, and termination signals. cDNAexpression vectors incorporating such elements include those describedby Okayama (Mol. Cell. Biol. 3:280-?, 1983).

[0118] The introduced nucleic acid molecule can be incorporated into aplasmid or viral vector capable of autonomous replication in therecipient host. Any of a wide variety of vectors may be employed forthis purpose. Factors of importance in selecting a particular plasmid orviral vector include: the ease with which recipient cells that containthe vector may be recognized and selected from those recipient cellswhich do not contain the vector; the number of copies of the vectorwhich are desired in a particular host; and whether it is desirable tobe able to “shuttle” the vector between host cells of different species.

[0119] Preferred prokaryotic vectors include plasmids such as thosecapable of replication in E. coil (such as, for example, pBR322, ColE1,pSC101, pACYC 184, nVX; “Molecular Cloning: A Laboratory Manual”, 1989,supra). Bacillus plasmids include pC194, pC221, pT127, and the like(Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, NY,pp. 307-329, 1982). Suitable Streptomyces plasmids include plJ101(Kendall et al., J. Bacteriol. 169:4177-4183, 1987), and streptomycesbacteriophages such as φC31 (Chater et al., In: Sixth InternationalSymposium on Actinomycetales Biology, Akademiai Kaido, Budapest,Hungary, pp. 45-54, 1986). Pseudomonas plasmids are reviewed by John etal. (Rev. Infect. Dis. 8:693-704, 1986), and Izaki (Jpn. J. Bacteriol.33:729-742, 1978).

[0120] Preferred eukaryotic plasmids include, for example, BPV,vaccinia, SV40, 2-micron circle, and the like, or their derivatives.Such plasmids are well known in the art (Botstein et al., Miami Wntr.Symp. 19:265-274, 1982; Broach, In: “The Molecular Biology of the YeastSaccharomyces: Life Cycle and Inheritance”, Cold Spring HarborLaboratory, Cold Spring Harbor, NY, p. 445-470, 1981; Broach, Cell28:203-204, 1982; Bollon et al., J. Ctin. Hematol. Oncol. 10:39-48,1980; Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3, GeneSequence Expression, Academic Press, NY, pp. 563-608, 1980).

[0121] Once the vector or nucleic acid molecule containing theconstruct(s) has been prepared for expression, the DNA construct(s) maybe introduced into an appropriate host cell by any of a variety ofsuitable means, i.e., transformation, transfection, conjugation,protoplast fusion, electroporation, particle gun technology, calciumphosphate-precipitation, direct microinjection, and the like. After theintroduction of the vector, recipient cells are grown in a selectivemedium, which selects for the growth of vector-containing cells.Expression of the cloned gene(s) results in the production of AUR1and/or AUR2 or fragments thereof. This can take place in the transformedcells as such, or following the induction of these cells todifferentiate (for example, by administration of bromodeoxyuracil toneuroblastoma cells or the like). A variety of incubation conditions canbe used to form the peptide of the present invention. The most preferredconditions are those which mimic physiological conditions.

[0122] V. Purified AUR1 and/or AUR2 Polypeptides

[0123] AUR1 and AUR2 are related serine/threonine kinases with shortN-terminal extensions. The Drosophila and yeast homologs appear to beinvolved in mitotic regulation. The human proteins appear to be involvedin cancer and/or other signal transduction disorders.

[0124] Primary sequence analysis of the human aur1 and aur2 genesreveals that they contain a highly conserved C-terminal protein kinasedomain with all the characteristic motifs of a serine/threonine kinase.In addition, the 73 to 129 amino acid N-terminal domains of human aur1and aur2 contain two distantly conserved motifs present in thenon-catalytic region of all aurora genes which may play a regulatoryrole or function as a substrate binding motif.

[0125] The first motif includes a 10 amino stretch, KENX₄PVK, termedAurora Box1. The second motif is centered around a 15 amino acidstretch, QX₉AQRVL, termed Aurora Box 2. Several potential serine andthreonine phosphorylation sites are also conserved among these proteinsincluding a protein kinase A phosphorylation motif, RRXT, in theactivation loop of the kinase, which suggests a regulatory pathwaysimilar to the cell cycle regulated CDC2/CDK-related proteins.

[0126] A temperature sensitive mutant of the yeast IPL1 gene consists ofa Thr to Ala substitution within the activation loop2, suggesting thatphosphorylation at this site may be biologically relevant. Additionalmutants in the yeast (Chan et al., Genetics 135:677-691, 1993) andDrosophila (Glover et al. Cell 81:95-105, 1995) homologues of aurorahave been mapped exclusively to the kinase domain, except for a singleDrosophila mutant that involves a mutation at Asp47 within theN-terminal Aurora Box2. These mutations result in abnormal nuclei,chromosome missegregation, and monopolar spindles.

[0127] Aur2 expression is primarily restricted to fetal liver, adulttestis, and thymus, suggestive of a normal role for these proteins inmeiotic division. Human Aur1 is also expressed at highest levels innormal testis and thymus, with a moderate level of expression in lungand small intestine. Very weak expression of Aur2 is also detected inbone marrow, lymph node, and spleen, and no expression is detected inall other adult tissues examined.

[0128] Additional studies demonstrate tight temporal regulation of thesetranscripts during mitosis (and Kimura et al., J. Biol. Chem.272:13766-13771, 1997). Both AUR1 and AUR2 appear to regulate nucleardivision, with disruption of their signaling resulting in polyploidcells. This phenotype is likely due to chromosomal missegregation, asseen with the yeast homologue IPL1.

[0129] AUR2 appears to play a role in cellular transformation. Ectopicexpression of activated AUR2, which can phosphorylate myelin basicprotin in vitro, confers a growth advantage to NIH3T3 cells in low serumas compared to wild-type AUR2, kinase inactive AUR2, and vector. Inaddition, only NIH3T3 cells expressing activated AUR2 grow largecolonies in soft agar thus resulting in anchorage-dependent growth.

[0130] However, in a rat1 fibroblast system, both the wild-type andactivated AUR2 (T288D) proteins were able to phosphorylate theartificial substrate, α-casein, over the levels observed in the vectorcontrol cell line. In addition, cells expressing the wild-type as wellas the activated mutant AUR2 formed colonies in soft agar, in contrastto the lack of growth by cells expressing the kinase inactive AUR2.

[0131] A variety of methodologies known in the art can be utilized toobtain the peptide of the present invention. The peptide may be purifiedfrom tissues or cells which naturally produce the peptide.Alternatively, the above-described isolated nucleic acid fragments couldbe used to express the AUR1 and/or AUR2 protein in any organism. Thesamples of the present invention include cells, protein extracts ormembrane extracts of cells, or biological fluids. The samples will varybased on the assay format, the detection method, and the nature of thetissues, cells or extracts used as the sample.

[0132] Any eukaryotic organism can be used as a source for the peptideof the invention, as long as the source organism naturally contains sucha peptide. As used herein, “source organism” refers to the originalorganism from which the amino acid sequence of the subunit is derived,regardless of the organism the subunit is expressed in and ultimatelyisolated from.

[0133] One skilled in the art can readily follow known methods forisolating proteins in order to obtain the peptide free of naturalcontaminants. These include, but are not limited to: size-exclusionchromatography, HPLC, ion-exchange chromatography, and immuno-affinitychromatography.

[0134] VI. An Antibody Having Binding Affinity To An AUR1 and/or AUR2Polypeptide And A Hybridoma Containing the Antibody

[0135] The present invention relates to an antibody having bindingaffinity to an AUR1 and/or AUR2 polypeptide. The polypeptide may havethe amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4, or afunctional derivative thereof, or at least 9 contiguous amino acidsthereof (preferably, at least 20, 30, 35, or 40 contiguous amino acidsthereof).

[0136] The present invention also relates to an antibody having specificbinding affinity to an AUR1 and/or AUR2 polypeptide. Such an antibodymay be isolated by comparing its binding affinity to an AUR1 and/or AUR2polypeptide with its binding affinity to other polypeptides. Those whichbind selectively to AUR1 and/or AUR2 would be chosen for use in methodsrequiring a distinction between AUR1 and/or AUR2 and other polypeptides.Such methods could include, but should not be limited to, the analysisof altered AUR1 and/or AUR2 expression in tissue containing otherpolypeptides.

[0137] The AUR1 and/or AUR2 proteins of the present invention can beused in a variety of procedures and methods, such as for the generationof antibodies, for use in identifying pharmaceutical compositions, andfor studying DNA/protein interaction.

[0138] The AUR1 and/or AUR2 peptide of the present invention can be usedto produce antibodies or hybridomas. One skilled in the art willrecognize that if an antibody is desired, such a peptide would begenerated as described herein and used as an immunogen. The antibodiesof the present invention include monoclonal and polyclonal antibodies,as well fragments of these antibodies, and humanized forms. Humanizedforms of the antibodies of the present invention may be generated usingone of the procedures known in the art such as chimerization or CDRgrafting. The present invention also relates to a hybridoma whichproduces the above-described monoclonal antibody, or binding fragmentthereof. A hybridoma is an immortalized cell line which is capable ofsecreting a specific monoclonal antibody.

[0139] In general, techniques for preparing monoclonal antibodies andhybridomas are well known in the art (Campbell, “Monoclonal AntibodyTechnology: Laboratory Techniques in Biochemistry and MolecularBiology,” Elsevier Science Publishers, Amsterdam, The Netherlands, 1984;St. Groth et al., J. Immunol. Methods 35:1-21, 1980). Any animal (mouse,rabbit, and the like) which is known to produce antibodies can beimmunized with the selected polypeptide. Methods for immunization arewell known in the art. Such methods include subcutaneous orintraperitoneal injection of the polypeptide. One skilled in the artwill recognize that the amount of polypeptide used for immunization willvary based on the animal which is immunized, the antigenicity of thepolypeptide and the site of injection.

[0140] The polypeptide may be modified or administered in an adjuvant inorder to increase the peptide antigenicity. Methods of increasing theantigenicity of a polypeptide are well known in the art. Such proceduresinclude coupling the antigen with a heterologous protein (such asglobulin or β-galactosidase) or through the inclusion of an adjuvantduring immunization.

[0141] For monoclonal antibodies, spleen cells from the immunizedanimals are removed, fused with myeloma cells, such as SP2/0-Agl4myeloma cells, and allowed to become monoclonal antibody producinghybridoma cells. Any one of a number of methods well known in the artcan be used to identify the hybridoma cell which produces an antibodywith the desired characteristics. These include screening the hybridomaswith an ELISA assay, western blot analysis, or radioimmunoassay (Lutz etal., Exp. Cell Res. 175:109-124, 1988). Hybridomas secreting the desiredantibodies are cloned and the class and subclass are determined usingprocedures known in the art (Campbell, “Monoclonal Antibody Technology:Laboratory Techniques in Biochemistry and Molecular Biology”, supra,1984).

[0142] For polyclonal antibodies, antibody containing antisera isisolated from the immunized animal and is screened for the presence ofantibodies with the desired specificity using one of the above-describedprocedures. The above-described antibodies may be detectably labeled.Antibodies can be detectably labeled through the use of radioisotopes,affinity labels (such as biotin, avidin, and the like), enzymatic labels(such as horse radish peroxidase, alkaline phosphatase, and the like)fluorescent labels (such as FITC or rhodamine, and the like),paramagnetic atoms, and the like. Procedures for accomplishing suchlabeling are well-known in the art, for example, see (Stemberger et al.,J. Histochem. Cytochem. 18:315, 1970; Bayer et al., Meth. Enzym.62:308-, 1979; Engval et al., Immunol. 109:129-, 1972; Goding, J.Immunol. Meth. 13:215-, 1976). The labeled antibodies of the presentinvention can be used for in vitro, in vivo, and in situ assays toidentify cells or tissues which express a specific peptide.

[0143] The above-described antibodies may also be immobilized on a solidsupport. Examples of such solid supports include plastics such aspolycarbonate, complex carbohydrates such as agarose and sepharose,acrylic resins and such as polyacrylamide and latex beads. Techniquesfor coupling antibodies to such solid supports are well known in the art(Weir et al., “Handbook of Experimental Immunology” 4th Ed., BlackwellScientific Publications, Oxford, England, Chapter 10, 1986; Jacoby etal., Meth. Enzym. 34, Academic Press, N.Y., 1974). The immobilizedantibodies of the present invention can be used for in vitro, in vivo,and in situ assays as well as in immunochromotography.

[0144] Furthermore, one skilled in the art can readily adapt currentlyavailable procedures, as well as the techniques, methods and kitsdisclosed above with regard to antibodies, to generate peptides capableof binding to a specific peptide sequence in order to generaterationally designed antipeptide peptides (Hurby et al., “Application ofSynthetic Peptides: Antisense Peptides”, In Synthetic Peptides, A User'sGuide, W. H. Freeman, NY, pp. 289-307, 1992; Kaspczak et al.,Biochemistry 28:9230-9238, 1989).

[0145] Anti-peptide peptides can be generated by replacing the basicamino acid residues found in the AUR1 and/or AUR2 peptide sequence withacidic residues, while maintaining hydrophobic and uncharged polargroups. For example, lysine, arginine, and/or histidine residues arereplaced with aspartic acid or glutamic acid and glutamic acid residuesare replaced by lysine, arginine or histidine.

[0146] VII. An Antibody Based Method And Kit For Detecting AUR1 and/orAUR2

[0147] Antibodies to AUR2 protein detect a protein of approximately 46kDa (the size of the AUR2 protein) in 2 primary human colon cancers, butnot in adjacent samples of normal tissue. Endogenous AUR2 is alsodetected in cultured tumor cell lines.

[0148] The present invention encompasses a method of detecting an AUR1and/or AUR2 polypeptide in a sample, comprising: (a) contacting thesample with an above-described antibody, under conditions such thatimmunocomplexes form, and (b) detecting the presence of said antibodybound to the polypeptide. In detail, the methods comprise incubating atest sample with one or more of the antibodies of the present inventionand assaying whether the antibody binds to the test sample. Alteredlevels of AUR1 and/or AUR2 in a sample as compared to normal levels mayindicate disease.

[0149] Conditions for incubating an antibody with a test sample vary.Incubation conditions depend on the format employed in the assay, thedetection methods employed, and the type and nature of the antibody usedin the assay. One skilled in the art will recognize that any one of thecommonly available immunological assay formats (such asradioimmunoassays, enzyme-linked immunosorbent assays, diffusion basedOuchterlony, or rocket immunofluorescent assays) can readily be adaptedto employ the antibodies of the present invention. Examples of suchassays can be found in Chard (“An Introduction to Radioimmunoassay andRelated Techniques” Elsevier Science Publishers, Amsterdam, TheNetherlands, 1986), Bullock et al. (“Techniques in Immunocytochemistry,”Academic Press, Orlando, Fla. Vol. 1, 1982; Vol. 2, 1983; Vol. 3, 1985),Tijssen (“Practice and Theory of Enzyme Immunoassays: LaboratoryTechniques in Biochemistry and Molecular Biology,” Elsevier SciencePublishers, Amsterdam, The Netherlands, 1985).

[0150] The immunological assay test samples of the present inventioninclude cells, protein or membrane extracts of cells, or biologicalfluids such as blood, serum, plasma, or urine. The test samples used inthe above-described method will vary based on the assay format, natureof the detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing protein extracts or membraneextracts of cells are well known in the art and can be readily beadapted in order to obtain a sample which is testable with the systemutilized.

[0151] A kit contains all the necessary reagents to carry out thepreviously described methods of detection. The kit may comprise: (i) afirst container means containing an above-described antibody, and (ii)second container means containing a conjugate comprising a bindingpartner of the antibody and a label. In another preferred embodiment,the kit further comprises one or more other containers comprising one ormore of the following: wash reagents and reagents capable of detectingthe presence of bound antibodies.

[0152] Examples of detection reagents include, but are not limited to,labeled secondary antibodies, or in the alternative, if the primaryantibody is labeled, the chromophoric, enzymatic, or antibody bindingreagents which are capable of reacting with the labeled antibody. Thecompartmentalized kit may be as described above for nucleic acid probekits. One skilled in the art will readily recognize that the antibodiesdescribed in the present invention can readily be incorporated into oneof the established kit formats which are well known in the art.

[0153] VIII. Isolation of Compounds Which Interact With AUR1 and/or AUR2

[0154] The present invention also relates to a method of detecting acompound capable of binding to an AUR1 and/or AUR2 polypeptidecomprising incubating the compound with AUR1 and/or AUR2 and detectingthe presence of the compound bound to AUR1 and/or AUR2. The compound maybe present within a complex mixture, for example, serum, body fluid, orcell extracts.

[0155] The present invention also relates to a method of detecting anagonist or antagonist of AUR1 and/or AUR2 activity or AUR1 and/or AUR2binding partner activity comprising incubating cells that produce AUR1and/or AUR2 in the presence of a compound and detecting changes in thelevel of AUR1 and/or AUR2 activity or AUR1 and/or AUR2 binding partneractivity. The compounds thus identified would produce a change inactivity indicative of the presence of the compound. The compound may bepresent within a complex mixture, for example, serum, body fluid, orcell extracts. Once the compound is identified it can be isolated usingtechniques well known in the art.

[0156] The present invention also encompasses a method of agonizing(stimulating) or antagonizing AUR1 and/or AUR2 associated activity in amammal comprising administering to said mammal an agonist or antagonistto AUR1 and/or AUR2 in an amount sufficient to effect said agonism orantagonism. A method of treating diseases in a mammal with an agonist orantagonist of AUR1 and/or AUR2 related activity comprising administeringthe agonist or antagonist to a mammal in an amount sufficient to agonizeor antagonize AUR1 and/or AUR2 associated functions is also encompassedin the present application.

[0157] IX. Transgenic Animals

[0158] A variety of methods are available for the production oftransgenic animals associated with this invention. DNA can be injectedinto the pronucleus of a fertilized egg before fusion of the male andfemale pronuclei, or injected into the nucleus of an embryonic cell(e.g., the nucleus of a two-cell embryo) following the initiation ofcell division (Brinster et al., Proc. Nat. Acad. Sci. USA 82: 4438-4442,1985). Embryos can be infected with viruses, especially retroviruses,modified to carry inorganic-ion receptor nucleotide sequences of theinvention.

[0159] Pluripotent stem cells derived from the inner cell mass of theembryo and stabilized in culture can be manipulated in culture toincorporate nucleotide sequences of the invention. A transgenic animalcan be produced from such cells through implantation into a blastocystthat is implanted into a foster mother and allowed to come to term.Animals suitable for transgenic experiments can be obtained fromstandard commercial sources such as Charles River (Wilmington, Mass.),Taconic (Germantown, N.Y.), Harlan Sprague Dawley (Indianapolis, Ind.),etc.

[0160] The procedures for manipulation of the rodent embryo and formicroinjection of DNA into the pronucleus of the zygote are well knownto those of ordinary skill in the art (Hogan et al., supra).Microinjection procedures for fish, amphibian eggs and birds aredetailed in Houdebine and Chourrout (Experientia 47: 897-905, 1991).Other procedures for introduction of DNA into tissues of animals aredescribed in U.S. Pat. No., 4,945,050 (Sandford et al., Jul. 30, 1990).

[0161] By way of example only, to prepare a transgenic mouse, femalemice are induced to superovulate. Females are placed with males, and themated females are sacrificed by CO₂ asphyxiation or cervical dislocationand embryos are recovered from excised oviducts. Surrounding cumuluscells are removed. Pronuclear embryos are then washed and stored untilthe time of injection. Randomly cycling adult female mice are pairedwith vasectomized males. Recipient females are mated at the same time asdonor females. Embryos then are transferred surgically. The procedurefor generating transgenic rats is similar to that of mice (Hammer etal., Cell 63:1099-1112, 1990).

[0162] Methods for the culturing of embryonic stem (ES) cells and thesubsequent production of transgenic animals by the introduction of DNAinto ES cells using methods such as electroporation, calciumphosphate/DNA precipitation and direct injection also are well known tothose of ordinary skill in the art (Teratocarcinomas and Embryonic StemCells, A Practical Approach, E. J. Robertson, ed., IRL Press, 1987).

[0163] In cases involving random gene integration, a clone containingthe sequence(s) of the invention is co-transfected with a gene encodingresistance. Alternatively, the gene encoding neomycin resistance isphysically linked to the sequence(s) of the invention. Transfection andisolation of desired clones are carried out by any one of severalmethods well known to those of ordinary skill in the art (E. J.Robertson, supra).

[0164] DNA molecules introduced into ES cells can also be integratedinto the chromosome through the process of homologous recombination(Capecchi, Science 244: 1288-1292, 1989). Methods for positive selectionof the recombination event (i.e., neo resistance) and dualpositive-negative selection (i.e., neo resistance and gancyclovirresistance) and the subsequent identification of the desired clones byPCR have been described by Capecchi, supra and Joyner et al. (Nature338: 153-156, 1989), the teachings of which are incorporated herein intheir entirety including any drawings. The final phase of the procedureis to inject targeted ES cells into blastocysts and to transfer theblastocysts into pseudopregnant females. The resulting chimeric animalsare bred and the offspring are analyzed by Southern blotting to identifyindividuals that carry the transgene. Procedures for the production ofnon-rodent mammals and other animals have been discussed by others(Houdebine and Chourrout, supra; Pursel et al., Science 244:1281-1288,1989; and Simms et al., Bio/Technology 6:179-183, 1988).

[0165] Thus, the invention provides transgenic, nonhuman mammalscontaining a transgene encoding an AUR1 and/or AUR2 polypeptide or agene effecting the expression of an AUR1 and/or AUR2 polypeptide. Suchtransgenic nonhuman mammals are particularly useful as an in vivo testsystem for studying the effects of introducing an AUR1 and/or AUR2polypeptide, or regulating the expression of an AUR1 and/or AUR2polypeptide (i.e., through the introduction of additional genes,antisense nucleic acids, or ribozymes).

[0166] A “transgenic animal” is an animal having cells that contain DNAwhich has been artificially inserted into a cell, which DNA becomes partof the genome of the animal which develops from that cell. Preferredtransgenic animals are primates, mice, rats, cows, pigs, horses, goats,sheep, dogs and cats. The transgenic DNA may encode for a human AUR1and/or AUR2 polypeptide. Native expression in an animal may be reducedby providing an amount of anti-sense RNA or DNA effective to reduceexpression of the receptor.

[0167] X. Gene Therapy

[0168] AUR2 protein expression in the human tumor cell line H1299 issignificantly down regulated in the presence of any one of threeantisense phosphothionate oligonucleotides (SEQ ID NO:30, SEQ ID NO:31and SEQ ID NO:32) which target specific regions of human aur2 mRNAtranscripts. When used in combination, oligonucleotides SEQ ID NO:30 andSEQ ID NO:32, and SEQ ID NO:31 and SEQ ID NO:32 reduce the expression ofAUR2 protein below the limit of detection. Treatment of H1299 cells withthe combination of SEQ ID NO:31 and SEQ ID NO:32 inhibited the growth ofthis tumor cell line, and induced apoptosis as measured by FACs.

[0169] AUR1 and/or AUR2 or its genetic sequences will also be useful ingene therapy (reviewed in Miller, Nature 357:455-460, 1992). Millerstates that advances have resulted in practical approaches to human genetherapy that have demonstrated positive initial results. The basicscience of gene therapy is described in Mulligan (Science 260:926-931,1993).

[0170] In one preferred embodiment, an expression vector containing theAUR1 and/or AUR2 coding sequence is inserted into cells, the cells aregrown in vitro and then infused in large numbers into patients. Inanother preferred embodiment, a DNA segment containing a promoter ofchoice (for example a strong promoter) is transferred into cellscontaining an endogenous aur1 and/or aur2 in such a manner that thepromoter segment enhances expression of the endogenous aur1 and/or aur2gene (for example, the promoter segment is transferred to the cell suchthat it becomes directly linked to the endogenous aur1 and/or aur2gene).

[0171] The gene therapy may involve the use of an adenovirus containingaur1 and/or aur2 cDNA targeted to a tumor, systemic AUR1 and/or AUR2increase by implantation of engineered cells, injection with aur1 and/oraur2 virus, or injection of naked aur1 and/or aur2 DNA into appropriatetissues.

[0172] Target cell populations may be modified by introducing alteredforms of one or more components of the protein complexes in order tomodulate the activity of such complexes. For example, by reducing orinhibiting a complex component activity within target cells, an abnormalsignal transduction event(s) leading to a condition may be decreased,inhibited, or reversed. Deletion or missense mutants of a component,that retain the ability to interact with other components of the proteincomplexes but cannot function in signal transduction may be used toinhibit an abnormal, deleterious signal transduction event.

[0173] Expression vectors derived from viruses such as retroviruses,vaccinia virus, adenovirus, adeno-associated virus, herpes viruses,several RNA viruses, or bovine papilloma virus, may be used for deliveryof nucleotide sequences (e.g., cDNA) encoding recombinant AUR1 and/orAUR2 protein into the targeted cell population (e.g., tumor cells).Methods which are well known to those skilled in the art can be used toconstruct recombinant viral vectors containing coding sequences(Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, N.Y., 1989; Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates and Wiley Interscience,N.Y., 1989). Alternatively, recombinant nucleic acid molecules encodingprotein sequences can be used as naked DNA or in a reconstituted systeme.g., liposomes or other lipid systems for delivery to target cells(e.g., Felgner et al., Nature 337:387-8, 1989). Several other methodsfor the direct transfer of plasmid DNA into cells exist for use in humangene therapy and involve targeting the DNA to receptors on cells bycomplexing the plasmid DNA to proteins (Miller, supra).

[0174] In its simplest form, gene transfer can be performed by simplyinjecting minute amounts of DNA into the nucleus of a cell, through aprocess of microinjection (Capecchi, Cell 22:479-88, 1980). Oncerecombinant genes are introduced into a cell, they can be recognized bythe cells normal mechanisms for transcription and translation, and agene product will be expressed. Other methods have also been attemptedfor introducing DNA into larger numbers of cells. These methods include:transfection, wherein DNA is precipitated with CaPO₄ and taken intocells by pinocytosis (Chen et al., Mol. Cell Biol. 7:2745-52, 1987);electroporation, wherein cells are exposed to large voltage pulses tointroduce holes into the membrane (Chu et al., Nucleic Acids Res.15:1311-26, 1987); lipofection/liposome fusion, wherein DNA is packagedinto lipophilic vesicles which fuse with a target cell (Felgner et al.,Proc. Natl. Acad. Sci. USA. 84:7413-7417, 1987); and particlebombardment using DNA bound to small projectiles (Yang et al., Proc.Natl. Acad. Sci. 87:9568-9572, 1990). Another method for introducing DNAinto cells is to couple the DNA to chemically modified proteins.

[0175] It has also been shown that adenovirus proteins are capable ofdestabilizing endosomes and enhancing the uptake of DNA into cells. Theadmixture of adenovirus to solutions containing DNA complexes, or thebinding of DNA to polylysine covalently attached to adenovirus usingprotein crosslinking agents substantially improves the uptake andexpression of the recombinant gene (Curiel et al., Am. J. Respir. Cell.Mol. Biol., 6:247-52, 1992).

[0176] As used herein “gene transfer” means the process of introducing aforeign nucleic acid molecule into a cell. Gene transfer is commonlyperformed to enable the expression of a particular product encoded bythe gene. The product may include a protein, polypeptide, anti-sense DNAor RNA, or enzymatically active RNA. Gene transfer can be performed incultured cells or by direct administration into animals. Generally genetransfer involves the process of nucleic acid contact with a target cellby non-specific or receptor mediated interactions, uptake of nucleicacid into the cell through the membrane or by endocytosis, and releaseof nucleic acid into the cytoplasm from the plasma membrane or endosome.Expression may require, in addition, movement of the nucleic acid intothe nucleus of the cell and binding to appropriate nuclear factors fortranscription.

[0177] As used herein “gene therapy” is a form of gene transfer and isincluded within the definition of gene transfer as used herein andspecifically refers to gene transfer to express a therapeutic productfrom a cell in vivo or in vitro. Gene transfer can be performed ex vivoon cells which are then transplanted into a patient, or can be performedby direct administration of the nucleic acid or nucleic acid-proteincomplex into the patient.

[0178] In another preferred embodiment, a vector having nucleic acidsequences encoding an AUR1 and/or AUR2 polypeptide is provided in whichthe nucleic acid sequence is expressed only in specific tissue. Methodsof achieving tissue-specific gene expression as set forth inInternational Publication No. WO 93/09236, filed Nov. 3, 1992 andpublished May 13, 1993.

[0179] In all of the preceding vectors set forth above, a further aspectof the invention is that the nucleic acid sequence contained in thevector may include additions, deletions or modifications to some or allof the sequence of the nucleic acid, as defined above.

[0180] In another preferred embodiment, a method of gene replacement isset forth. “Gene replacement” as used herein means supplying a nucleicacid sequence which is capable of being expressed in vivo in an animaland thereby providing or augmenting the function of an endogenous genewhich is missing or defective in the animal.

[0181] XI. Compounds that Modulate the Function of AUR1 and/or Aur2Proteins

[0182] In an effort to discover novel treatments for diseases,biomedical researchers and chemists have designed, synthesized, andtested molecules that inhibit the function of protein kinases. Somesmall organic molecules form a class of compounds that modulate thefunction of protein kinases. Examples of molecules that have beenreported to inhibit the function of protein kinases include, but are notlimited to, bis monocyclic, bicyclic or heterocyclic aryl compounds (PCTWO 92/20642, published Nov. 26, 1992 by Maguire et al.),vinylene-azaindole derivatives (PCT WO 94/14808, published Jul. 7, 1994by Ballinari et al.), 1-cyclopropyl-4-pyridylquinolones (U.S. Pat. No.5,330,992), styryl compounds (U.S. Pat. No. 5,217,999),styryl-substituted pyridyl compounds (U.S. Pat. No. 5,302,606), certainquinazoline derivatives (EP Application No. 0 566 266 A1), seleoindolesand selenides (PCT WO 94/03427, published Feb. 17, 1994 by Denny etal.), tricyclic polyhydroxylic compounds (PCT WO 92/21660, publishedDec. 10, 1992 by Dow), and benzylphosphonic acid compounds (PCT WO91/15495, published Oct. 17, 1991 by Dow et al). The compounds that cantraverse cell membranes and are resistant to acid hydrolysis arepotentially advantageous therapeutics as they can become highlybioavailable after being administered orally to patients. However, manyof these protein kinase inhibitors only weakly inhibit the function ofprotein kinases. In addition, many inhibit a variety of protein kinasesand will therefore cause multiple side-effects as therapeutics fordiseases.

[0183] Some indolinone compounds, however, form classes of acidresistant and membrane permeable organic molecules. WO 96/22976,published Aug. 1, 1996 by Ballinari et al. describes hydrosolubleindolinone compounds that harbor tetralin, naphthalene, quinoline, andindole substituents fused to the oxindole ring. These bicyclicsubstituents are in turn substituted with polar moieties includinghydroxylated alkyl, phosphate, and ether moieties. U.S. patentapplication Ser. Nos. 08/702,232, filed Aug. 23, 1996, entitled“Indolinone Combinatorial Libraries and Related Products and Methods forthe Treatment of Disease” by Tang et al. (Lyon & Lyon Docket No.221/187) and 08/485,323, filed Jun. 7, 1995, entitled“Benzylidene-Z-Indoline Compounds for the Treatment of Disease” by Tanget al. (Lyon & Lyon Docket No. 223/298) and International PatentPublication WO 96/22976, published Aug. 1, 1996 by Ballinari et al., allof which are incorporated herein by reference in their entirety,including any drawings, describe indolinone chemical libraries ofindolinone compounds harboring other bicyclic moieties as well asmonocyclic moieties fused to the oxindole ring. Applications 08/702,232,filed Aug. 23, 1996, entitled “Indolinone Combinatorial Libraries andRelated Products and Methods for the Treatment of Disease” by Tang etal. (Lyon & Lyon Docket No. 221/187), 08/485,323, filed Jun. 7, 1995,entitled “Benzylidene-Z-Indoline Compounds for the Treatment of Disease”by Tang et al. (Lyon & Lyon Docket No. 223/298), and WO 96/22976,published Aug. 1, 1996 by Ballinari et al. teach methods of indolinonesynthesis, methods of testing the biological activity of indolinonecompounds in cells, and inhibition patterns of indolinone derivatives.

[0184] Other examples of substances capable of modulating AUR1 and/orAUR2 activity include, but are not limited to, tyrphostins,quinazolines, quinoxolines, and quinolines.

[0185] The quinazolines, tyrphostins, quinolines, and quinoxolinesreferred to above include well known compounds such as those describedin the literature. For example, representative publications describingquinazoline include Barker et al., EPO Publication No. 0 520 722 A1;Jones et al., U.S. Pat. No. 4,447,608; Kabbe et al., U.S. Pat. No.4,757,072; Kaul and Vougioukas, U.S. Pat. No. 5,316,553; Kreighbaum andComer, U.S. Pat. No. 4,343,940; Pegg and Wardleworth, EPO PublicationNo. 0 562 734 A1; Barker et al., Proc. of Am. Assoc. for Cancer Research32:327 (1991); Bertino, J. R., Cancer Research 3:293-304 (1979);Bertino, J. R., Cancer Research 9(2 part 1):293-304 (1979); Curtin etal., Br. J. Cancer 53:361-368 (1986); Fernandes et al., Cancer Research43:1117-1123 (1983); Ferris et al. J. Org. Chem. 44(2):173-178; Fry etal., Science 265:1093-1095 (1994); Jackman et al., Cancer Research51:5579-5586 (1981); Jones et al. J. Med. Chem. 29(6):1114-1118; Lee andSkibo, Biochemistry 26(23):7355-7362 (1987); Lemus et al., J. Org. Chem.54:3511-3518 (1989); Ley and Seng, Synthesis 1975:415-522 (1975);Maxwell et al., Magnetic Resonance in Medicine 17:189-196 (1991); Miniet al., Cancer Research 45:325-330 (1985); Phillips and Castle, J.Heterocyclic Chem. 17(19):1489-1596 (1980); Reece et al., CancerResearch 47(11) :2996-2999 (1977); Sculier et al., Cancer Immunol. andImmunother. 23:A65 (1986); Sikora et al., Cancer Letters 23:289-295(1984); Sikora et al., Analytical Biochem. 172:344-355 (1988); all ofwhich are incorporated herein by reference in their entirety, includingany drawings.

[0186] Quinoxaline is described in Kaul and Vougioukas, U.S. Pat. No.5,316,553, incorporated herein by reference in its entirety, includingany drawings.

[0187] Quinolines are described in Dolle et al., J. Med. Chem.37:2627-2629 (1994); MaGuire, J. Med. Chem. 37:2129-2131 (1994); Burkeet al., J. Med. Chem. 36:425-432 (1993); and Burke et al. BioOrganicMed. Chem. Letters 2:1771-1774 (1992), all of which are incorporated byreference in their entirety, including any drawings.

[0188] Tyrphostins are described in Allen et al., Clin. Exp. Immunol.91:141-156 (1993); Anafi et al., Blood 82:12:3524-3529 (1993); Baker etal., J. Cell Sci. 102:543-555 (1992); Bilder et al., Amer. Physiol. Soc.pp. 6363-6143:C721-C730 (1991); Brunton et al., Proceedings of Amer.Assoc. Cancer Rsch. 33:558 (1992); Bryckaert et al., Experimental CellResearch 199:255-261 (1992); Dong et al., J. Leukocyte Biology 53:53-60(1993); Dong et al., J. Immunol. 151(5):2717-2724 (1993); Gazit et al.,J. Med. Chem. 32:2344-2352 (1989); Gazit et al., “J. Med. Chem.36:3556-3564 (1993); Kaur et al., Anti-Cancer Drugs 5:213-222 (1994);Kaur et al., King et al., Biochem. J. 275:413-418 (1991); Kuo et al.,Cancer Letters 74:197-202 (1993); Levitzki, A., The FASEB J. 6:3275-3282(1992); Lyall et al., J. Biol. Chem. 264:14503-14509 (1989); Peterson etal., The Prostate 22:335-345 (1993); Pillemer et al., Int. J. Cancer50:80-85 (1992); Posner et al., Molecular Pharmacology 45:673-683(1993); Rendu et al., Biol. Pharmacology 44(5):881-888 (1992); Sauro andThomas, Life Sciences 53:371-376 (1993); Sauro and Thomas, J. Pharm. andExperimental Therapeutics 267(3):119-1125 (1993); Wolbring et al., J.Biol. Chem. 269(36):22470-22472 (1994); and Yoneda et al., CancerResearch 51:4430-4435 (1991); all of which are incorporated herein byreference in their entirety, including any drawings.

[0189] Other compounds that could be used as modulators includeoxindolinones such as those described in U.S. patent application Ser.No. 08/702,232 filed Aug. 23, 1996, incorporated herein by reference inits entirety, including any drawings.

EXAMPLES

[0190] The examples below are non-limiting and are merely representativeof various aspects and features of the present invention. The examplesbelow demonstrate the isolation, and characterization of the novelproteins AUR1 and AUR2.

Example 1 Cloning of Aur1 and Aur2 and Structural Motifs

[0191] Materials and Methods:

[0192] Molecular Cloning

[0193] Total RNAs were isolated using the Guanidine Salts/Phenolextraction protocol of Chomczynski and Sacchi (Anal. Biochem.162:156-159, 1987) from normal human prostate, duodenum, ovary, liver,pituitary, brain, thymus, and salivary gland, from human HEPM cells(palatal mesenchyme), from primary human Wilm's tumor and ovariancarcinoma, and from human tumor cell lines originating from colon/rectum(HT29, SW480, SW1463, SW1417, SW837, SW948, SW620, SW403, SW1116, T84,HTC15, LS123, and Caco-2), kidney (CaKi-1, CaKi-2), liver (SK-HEP-1),pancreas (HS766T, ASPC, Capan-1), and breast (MCF7).

[0194] These RNAs were used as templates to generate single-strandedcDNAs using the Superscript Preamplification System for First StrandSynthesis kit purchased from GibcoBRL (Life Technologies, U.S.A.; Gerardet al. 1989, FOCUS 11, 66) under conditions recommended by manufacturer.A typical reaction used 10 μg total RNA or 2 μg poly(A)⁺ RNA with 1.5 μgoligo(dT)₁₂₋₁₈ in a reaction volume of 60 μL. The product was treatedwith RNaseH and diluted to 100 μL with H₂O. For subsequent PCRamplification, 1-4 μl of these sscDNAs were used in each reaction.

[0195] Oligonucleotides were synthesized on an Applied Biosystems 394DNA synthesizer using established phosphoramidite chemistry and wereused unpurified after precipitation with ethanol. The degenerateoligonucleotide primers are: A   = 5′-GARTTYGGNGARGTNTTYYTNGC-3′ (SEQ IDNO:16) (sense) and DVW = 5′-AGNACNCCRAANGCCCACACRTC-3′ (SEQ ID NO:17)(antisense).

[0196] These primers were derived from the peptide sequences EFGEVFLA(SEQ ID NO:18) (sense strand from kinase subdomain I) and DVW(A/S)FGVL(SEQ ID NO:29; antisense strand from kinase subdomain IX), respectively.Degenerate nucleotide residue designations are: N=A, C, G, or T; R=A orG; and Y=C or T. Using CCK4 as a template, these primers produce aproduct of 567 bp.

[0197] A PCR reaction was performed using Primers A and DVW applied tothe single-stranded sources listed above. The primers were added at afinal concentration of 5 μM each to a mixture containing 10 mM Tris-HCl(pH8.3), 50 mM KCl, 1.5 mM MgCl₂, 200 μM each deoxynucleosidetriphosphate, 0.001% gelatin, and 1.5 U AmpliTaq DNA Polymerase(Perkin-Elmer/Cetus), and 1-4 μL cDNA. Following 3 min denaturation at95° C., the cycling conditions were 94° C. for 30 s, 37° C. for 1 min, a2 min ramp to 72° C., and 72° C. for 1 min for the first 3 cycles,followed by 94° C. for 30 s, 50° C. for 1 min, and 72° C. for 1 min 45 sfor 35 cycles. PCR fragments migrating at between 500-600 bp wereisolated from 2% agaorse gels using GeneClean (Bio101), and T-A clonedinto the pCRII vector (Invitrogen Corp. U.S.A.) according to themanufacturer's protocol.

[0198] Colonies were selected for mini plasmid DNA-preparations usingQiagen columns and the plasmid DNAs were sequenced using a cyclesequencing dye-terminator kit with AmpliTaq DNA Polymerase, FS (ABI,Foster City, Calif.). Sequencing reaction products were run on an ABIPrism 377 DNA Sequencer, and analyzed using the BLAST alignmentalgorithm (Altschul et al., J. Mol. Biol. 215:403-410, ?). A novel clone(#43-43) was isolated by PCR with primers A and DVW on single-strandedcDNA from human embryonic palatal mesenchyme (HEPM or CRL1486) as atemplate. This clone was subsequently designated as a fragment of humanaur1.

[0199] A lambda ZapII (Stratagene Cloning Systems, La Jolla, Calif.)cDNA library was constructed using mRNA from a pool of pancreaticcarcinoma cell lines as a template for first strand cDNA synthesis.Phage were screened on nitrocellulose filters with the random primed³²P-labeled insert from p43-43 encoding human aur1 at 2×10⁶ cpm/mL inhybridization buffer containing 6×SSC, 1×Denhardt's reagent, 0.1% SDS,with 0.1 mg/mL denatured, fragmented salmon sperm DNA. After overnighthybridization at 65° C., filters were washed in 0.1×SSC, 0.1% SDS at 65°C. Full length cDNA clones were sequenced on both strands using manualsequencing with T7 polymerase and oligonucleotide primers (Tabor et al.,Proc. Natl. Acad. Sci. U.S.A. 84: 4767-4771, 1987).

[0200] Southern Blot Analysis

[0201] Genomic DNA was isolated from a variety of transformed humanlines (CaCO2, HTC15, LS147T, SKCO4, SW480, SW403, SW620, SW948, SW1417,SW1116, MCF7, BT474) using standard procedures (Maniatis et al. supra).Cells were trypsinized, washed with PBS and resuspended at ˜10⁸ cells/mLin Digestion buffer (100 mM NaCl, 10 mM Tris pH8, 25 mM EDTA, pH8, 0.5%SDS, 0.1 mg/mL proteinase K). Cells were lysed by incubation at 50° C.for 12 hours, followed by extraction with phenol/chloroform andprecipitated with an equal volume of 7.5 M ammonium acetate and 100%EtOH. DNAs were resuspended in TE buffer. Approximately 20 μg genomicDNA was digested with HindIII or XhoII at 37° C. for at least 4 hoursbefore fractionation on 1% agarose gels. The DNA fragments weretransferred to nitrocellulose membranes by the capillary transfer method(Southern, J. Mol. Biol. 98:503-, 1975) and hybridized with human aur1and aur2-specifc probes as described for Northern Blot analysis below.DNAs were restricted with HindIII since both aur1 and aur2 cDNAs containa single site for this restriction enzyme.

[0202] Results:

[0203] In order to identify homologues of CCK4, a receptor thatrepresents a distinct family of tyrosine kinases, degenerate primers toconserved sequences within kinase subdomains I and IX of ROS and theTRK-family of receptor tyrosine kinases were designed, since multiplealignments suggested CCK4 was most closely related these receptors.Subdomain I is at the N-terminus of the kinase domain and contains theconsensus motif GXGXXGXV (SEQ ID NO:26) which is involved in anchoringATP to the catalytic unit of all classes of kinases. Subdomain IXcontains a nearly invariant Asp which acts to stabilize the catalyticloop by bonding to residues in subdomain VIB. Based on comparison of allknown protein kinases, degenerate oligonucleotide primers to subdomainsI and IX that would pick up only CCK4 and its chicken homologue KLG byPCR were designed.

[0204] Degenerate primers A and DVW were designed based on conservedresidues within the kinase domain of CCK4, to use for identification ofnovel kinases using polymerase chain reaction (PCR). When applied toHEPM cell sscDNA as a template, multiple copies of CCK4 were isolated aswell as a novel DNA fragment (43-43) of 567 bp with homology to otherkinases. The novel sequence was most similar to Drosophila aurora kinase(GeneBank Accession #X83465) and the clone was designated human aur1.

[0205] The aur1 probe was used to screen a cDNA library constructed fromhuman pancreatic cancer cell line mRNA to isolate overlapping clonesspanning the complete open reading frame of aur1. Of multiple clonesisolated, seven corresponded to human aur1. Two additional faintlyhybridizing clones were also isolated during this screen and sequenceanalysis revealed they corresponded to a related, yet distinct kinase,which we designated human aur2.

[0206] Aur1 showed a single 4.3 kb band of equal intensity from allsources suggesting it is a single copy, non-rearranged gene in themultiple tumor types assayed. However, under low stringency conditions,it was possible to detect 1.3 kb and 3.2 kb SacI fragments which weaklyhybridize to the aur1 probe. Aur2 showed bands at 7.0 kb and 4.3 kb anda faint higher molecular weight band at ˜10 kb from all sources. Thesedata suggest aur2 is also a single copy gene. The multiple bands seen onblots probed with aur2 are likely due to the fact that a full lengthcDNA probe was used.

[0207] The complete sequences of human aur1 and aur2 were determinedfrom full length clones of each, isolated from the human pancreaticcarcinoma library, from normal human duodenum, and from the partialhuman aur1 isolated from HEPM cells.

[0208] The 1,244 bp human aur1 nucleotide sequence is shown in SEQ IDNO:1 and contains a single open reading frame encoding a polypeptide of344 amino acids. The AUR1 coding region is flanked by a 54 nucleotide5′-untranslated region and a 132 nucleotide 3′-untranslated regionending with a poly(A) tail.

[0209] The 2,198 bp human aur2 nucleotide sequence is shown in SEQ IDNO:2 and contains a single open reading frame encoding a polypeptide of403 amino acids. The AUR2 coding region is flanked by a 200 nucleotide5′-untranslated region and a 768 nucleotide 3′-untranslated region.

[0210] The aur1 and aur2 cDNAs were sequenced from both a humanpancreatic tumor and normal human duodenum, with no sequence differencesobserved except some probably polymorphic sites. These ambiguitiesinclude: cDNA nucleotide Comment aur1 1174 one clone has poly A inserted873 T in all duodenal clones, C in pancreatic tumor 469 T in one clone,C in all others 848 G in one clone, A in all others - changes amino acidE to G 1097 G in one clone, T in 2 others 956 G in one clone, A in 4others 29 Splice to 103 in 5 clones, no splice (as sown in 5 clones aur2349 T in 1 cline, C in multiple others (change amino acid P to L) 369 Ain 3 clones, G in multiple others (change AA V to I)

[0211] The C-terminal portions AUR1 and AUR2 conserve all 12 subdomainscharacteristic of eukaryotic protein kinases. The AUR1 and AUR2 kinasedomains are preceded by a N-terminal domain of 74 and 130 amino acids,respectively. Comparison of the aur1 and aur2 nucleotide and deducedamino acid sequences (SEQ ID NO:3 or SEQ ID NO:4) with the available DNAand protein sequence databases indicated that they are unique with theexception of several EST sequences sharing high sequence identity. Theydo however have striking homology in both the N-terminal and catalyticdomains with the drosophila aurora and Saccharomyces cerevisiae IPL1genes. Furthermore, two unpublished database entries are likely to beclose homologues from Xenopus laevis (p46APK-GB accession #Z17206 andp46BPK-GB accession #Z17207).

[0212] The N-terminal domains of aurora from human, frog, Drosophila,and yeast share limited sequence identity. Comparison of the catalyticdomains of these proteins reveals AUR1 shares 70% amino acid identitywith AUR2, 61% with the Drosophila aurora, and 45% with the yeast IPL1gene. AUR2 kinase shares 60% amino acid identity with the Drosophilaprotein and 45% identity to yeast IPL1. Both AUR1 and AUR2 share lessthan 45% homology with all other known mammalian kinases (the closestbeing cAMP-dependent protein kinase A) suggesting they are homologues ofthese drosophila and yeast kinases.

[0213] AUR1 and AUR2 both contain a cAMP-dependent protein kinasephosphorylation site (THR232 of AUR1 and THR288 of AUR2) that isconserved in the drosophila and yeast homologues and is a knownregulatory site in the cyclin-dependent kinase p34cdc2. AUR2 contains anadditional PKA-site at SER342. Both proteins also have multiple Caseinkinase II (five and six for AUR1 and AUR2) and protein kinase C (fourand ten for AUR1 and AUR2) phosphorylation sites. AUR2 also has a singletyrosine phosphorylation consensus site at TYR334 that is also conservedwith the Drosophila aurora, but is not present in AUR1 or yeast IPL1.

[0214] Natural mutants of the drosophila aurora AUR_dm and yeast IPL1gene result in asymmetric nuclear division leading to chromosomemissegregation, and atypical, monopolar spindles. This phenotype appearsto result from a failure of centrosome separation. The associatedmicrotubule architecture appear unaffected. Natural mutants in bothdrosophila and yeast target amino acid residues that are strictlyconserved between human AUR1 and AUR2, further supporting they may befunctional homologues. The corresponding residues in AUR1 that are foundin natural mutants of AUR_dm or IPL1 are GLU125, THR232, PRO312, HIS324.All of these mutations are within the catalytic domain, and notably, onerepresents the conserved PKA-phosphorylation site. An additionalmutation in AUR_dm at ASP47 is at a non-conserved residue in theN-terminal domain.

[0215] These findings suggest the catalytic activity may indeed play acentral role in the biology of centrosome replication or segregation inlower organisms, and suggest that human AUR1 and AUR2 may play acomplementary role in mammalian cells.

Example 2 AUR1 and AUR2 Expression in Human Tissues

[0216] Materials and Methods:

[0217] Northern Blot Analysis

[0218] Northern blots containing 2 μg poly A+RNA per lane from 16different adult human tissues (spleen, thymus, prostate, testis, ovary,small intestine, colonic mucosa, heart, brain, placenta, lung, liver,skeletal muscle, kidney, pancreas, and peripheral blood leukocytes),four different human fetal tissues (brain, lung, liver, and kidney), and8 human cancer cell lines (HL60, HeLa, K-562, MOLT-4, Raji, SW480, andG361) on a charge-modified nylon membrane were obtained from Clontech(Palo Alto, Calif.). Additional Northern blots were prepared by running10 μg total RNA isolated from human tumor cell lines on a denaturingformaldehyde 1.2% agarose gel and transferring to nylon membranes.

[0219] Filters were hybridized with random primed [³²P]dCTP-labeledprobes synthesized from either the 527 bp insert from human aur1 clone43-43 or the 1162 bp EcoRI fragment from pSG20, and either the 1 kbEcoRI fragment of human aur2 clone 11-1A or the 1257 bp BamHI-Not Ifragment from pS621. Hybridization was performed at 60° C. overnight in6×SSC, 0.1% SDS, 1×Denhardt's solution, 100 mg/mL denatured herringsperm DNA with 1-2×10⁶ cpm/mL of ³²P-labeled DNA probes. The filterswere washed in 0.1×SSC/0.1% SDS, 65° C., and exposed overnight on KodakXAR-2 film.

[0220] Semi-Ouantitative PCR Detection of aur1

[0221] RNA was isolated from a variety of human cell lines, fresh frozentissues, and primary tumors. Single stranded cDNA was synthesized from10 mg of each RNA as described above using the SuperscriptPreamplification System (GibcoBRL). These single strand templates werethen used in a 35 cycle PCR reaction with two aur1-specificoligonucleotides (3476: 5′-TTTGGCTCGGGAGAAGAAAAGCCAT-3′ (SEQ ID NO:19),and 3506: 5′-CAATCATCTCTGGGGGCAGGTAGT-3′) (SEQ ID NO:20). Reactionproducts were electrophoresed on 2% agarose gels, stained with ethidiumbromide and photographed on a UV light box. The relative intensity ofthe ˜475 bp aur1-specific bands were estimated for each sample.

[0222] Results:

[0223] A single aur1 mRNA transcript of approximately 1.4 kb wasidentified, and was found to be most abundant in the thymus and smallintestine with weak signals from testis, ovary, colon, placenta, andspleen. Prostate and peripheral blood lymphocytes were negative. Humanfetal liver and kidney were also positive, with a weaker signal in fetallung and no expression in fetal brain (Table)

[0224] A similar analysis of human aur2 expression showed a morerestricted expression profile. A single 2.4 kb aur2 transcript wasdetected strongly in the adult testis and thymus, and weakly in heart,placenta, skeletal muscles and in fetal liver and kidney whereas theother normal tissue sources were negative (see Table).

[0225] The aur1 mRNA expression profile in several primary tumors andmultiple cell lines of diverse neoplastic origin were determined byNorthern analysis and by the semi-quantitative PCR assay using primersfrom sequences in the aur1 kinase domain. The results are included inthe Table. Aur1 transcripts were detected in every tumor line assayedwith the highest expression in several human colon cancer cell lines(SW480, Colo320, SW620, SW1417, Caco2, SW12417) and in lung carcinoma(Calu3), breast carcinoma (T47D, MCF7), Melanoma (A375), Kidneycarcinoma (CaKi-1, CaKi-2), liver carcinoma (SK-HEP-1), and neuraltumors (SF767, T98G). Lesser expression of aur1 was seen in other coloncarcinomas (HTC15, T84, SW948, SW1116, HT29), neural tumors (Daoy),Ovarian carcinoma (Ovcar3, Primary tumor), pancreatic carcinoma(HS766T), and a primary kidney tumor.

[0226] The aur2 expression profile in tumor cell lines was strikinglymore restricted than that of aur1. Strong expression of aur2 wasdetected only in colon carcinoma cell lines (Caco2, SW480, SW1417,Sw620) whereas weak signals were seen in other colon (HTC15, Colo320),Breast (T47D, MCF7) and lung (Calu3) tumor cell lines. Several othertumor lines had no detectable aur2 transcripts. AUR1 and AUR2 NORTHERNANALYSIS IN HUMAN NORMAL TISSUE AND CANCER CELLS Cell type Origin AUR 1AUR 2 Thymus Normal tissue 5 4 Fetal liver Normal tissue 4 2 Fetalkidney Normal tissue 4 1 Lung Normal tissue 3 0 Duodenum Normal tissue 21 Colon Normal tissue 2 0 Fetal lung Normal tissue 2 0 Ovary Normaltissue 2 0 Testis Normal tissue 2 2 Brain Normal tissue 0 0 CerebellumNormal tissue 0 0 Salivary gland Normal tissue 0 0 Heart Normal tissue 00 Liver Normal tissue 0 0 Pancreas Normal tissue 0 0 Kidney Normaltissue 0 0 Spleen Normal tissue 0 0 Stomach Normal tissue 0 0 UterusNormal tissue 0 0 Prostate Normal tissue 0 0 Skeletal muscle Normaltissue 0 0 Fetal brain Normal tissue 0 0 PBLs Normal tissue 0 0 Salivarygland Normal tissue 0 0 Placenta Normal tissue 0 0 SF-268 CNS tumor 4 NDCCRF-CEM Leukemia 4 ND K-562 Leukemia 4 ND HCC-2998 Colon tumor 4 NDSW620 Colon tumor 4 2 KM-12 Leukemia 4 ND MCF7/ADR-RES Breast tumor 4 2MDA-N Breast tumor 4 ND BT-549 Breast tumor 4 ND SW480 Colon tumor 4 4SW48 Colon tumor 4 ND Calu-3 Lung tumor 4 ND Calu3 Lung tumor 4 2 T47DBreast tumor 4 2 A375 Melanoma 4 0 SF767 CNS tumor 4 0 SW1417 Colontumor 4 4 CaKi2 Kidney tumor 4 0 CaKi1 Kidney tumor 4 0 Caco2 Colontumor 4 4 SW1417 Colon tumor 4 0 T98G CNS tumor 4 0 SF-539 CNS tumor 3ND SK-MEL-2 Melanoma 3 ND SK-MEL-5 Melanoma 3 ND R-48 Gastric tumor 3 NDRF-1 Gastric tumor 3 ND SW948 Colon tumor 3 ND AGS Gastric tumor 3 NDHFL1 Normal lung 3 ND OVCAR-8 Ovarian tumor 2 ND HT-29 Colon tumor 2 NDMDA-MB-231 Breast tumor 2 ND MDA-MB-435 Breast tumor 2 ND SK-MEL-5Melanoma 2 ND Kato-3 Gastric tumor 2 ND Colo 205 Colon tumor 2 ND Colo320DM Colon tumor 2 2 WiDr Colon tumor 2 ND HT-29 Colon tumor 2 NDSNU-C2B Colon tumor 2 ND HTC15 Colon tumor 2 2 T84 Colon tumor 2 0 SW948Colon tumor 2 0 Daoy CNS tumor 2 0 OVCAR3 Ovary tumor 2 0 HS766TPancreas tumor 2 0 SW1116 Colon tumor 2 0 Wilms tumor Kidney tumor 2 0UO-31 Renal tumor 0 ND

Example 3 Recombinant Expression of Aur1 and Aur2

[0227] Materials and Methods:

[0228] Expression Vector Construction

[0229] Expression constructs were generated by PCR-assisted mutagenesisin which the entire coding domains of aur1 and aur2 were tagged on theircarboxy-terminal ends with the hemophilus influenza hemaglutinin (HA)epitope YPYDVPDYAS (SEQ ID NO:21)(Pati, Gene 114:285-288, 1992). Theseconstructs were introduced into two mammalian expression vectors: pLXSN(Miller et al., Biotechniques 7:980-988, 1989) for the generation ofvirus producing lines; and pRK5 for transient expression analysis.Inserts were designed to be flanked by unique BamHI and NotI sites andwere cloned directly into pLXSN or pRK5 at the 5′-BamHI and 3′-NotIsites.

[0230] The BamHI-NotI full length aur1 and aur2 constructs were alsoligated into pRS316 (Liu et al, Genetics 132:665-673, 1992). This vectorcontains a galactose-inducible promoter in a centromeric shuttle vectorfor expression in Saccharomyces cerevisiae. These are to assess if thehuman genes can complement the related temperature sensitive yeast IPL1mutant, which is closely related to aur1. In addition, fusion constructscontaining the N-terminal domain of yeast IPL1 fused to the C-terminalkinase domains of aur1 and aur2 were generated. These were produced byinsertion of an artificial ClaI site at the 5′ end of the kinase domainsof the kinases, at the conserved Asp-Asp-Phe-Glu sequence.

[0231] Dominant negative aur1 and aur2 constructs were also made in bothPLXSN and pRK5 by mutation of the invariant Lys (amino acid positions106 and 162 in AUR1 and AUR2, respectively) to an Met by PCRmutagenesis. The constructs are termed AUR1KM and AUR2KM. Constitutivelyactive forms of AUR1 and AUR2 were generated by mutation of the DNAheading the encoding the phosphorylation site (232 and 288) to an Aspresulting in AUR1TD and AUR2TD.

[0232] Expression constructs in both pLXSN and pRK5 were also madecontaining just the N-terminal, non-catalytic domain of AUR1 and AUR2.These were generated by PCR from the parental constructs and contain theN-terminal 77 amino acids of AUR1 and 132 amino acids of AUR2.

[0233] The entire aur1 and aur2 open reading frames (no HA-tag)excluding the initiating methionines were generated by PCR and ligatedinto pGEX vector for bacterial production of GST-fusion proteins forimmunization of rabbits for antibody production.

[0234] Generation of Virus Producing AUR Cell Lines

[0235] To generate high-titer virus stocks, pLXSN recombinant constructscontaining either aur1 or aur2 genes were transfected into anamphotropic helper cell line PA317 using CaCl₂-mediated transfection.After selection on G418, the cells were plated on normal media withoutG418 (500 ug/ml). Supernatants from resistant cells were used to infectthe ecotropic helper cell line GP+E86, and cells again selected on G418.Resistant cells were again taken off G418, and the supernatantsharvested every 8-12 hours and pooled as virus stock (Redemann et al.,Mol. Cell. Biol. 12, 491-498, 1992). Viral stock titers were typically˜10⁶/mL.

[0236] Retroviral Infection of NIH-3T3 Cells with Aur1 and/or Aur2

[0237] NIH-3T3, and BALB/3T3 cells were grown in 100 mm plates with DMEM(Gibco) containing 10% fetal calf serum (FCS). The cells weresuperinfected with the aur1 and aur2 retrovirus by adding approximately3 mL viral supernatant to 15 mL culture media for approximately 24hours. Cells expressing the retroviral constructs were then selected bygrowth in DMEM/10% FCS supplemented with 500 μg/mL G418.

[0238] Transient Expression of Aur1 and/or Aur2 in Mammalian Cells

[0239] The pRK5 expression plasmids (10 μg DNA/100 mm plate) containingthe HA-tagged aur1 and aur2 genes were introduced into COS and 293 cellswith lipofectamine (Gibco BRL). After 72 hours, the cells were harvestedin 0.5 mL solubilization buffer (20 mM HEPES pH7.35, 150 mM NaCl, 10%glycerol, 1% Triton X-100, 1.5 mM MgCl₂, 1 mM EGTA, 2 mMphenylmethylsulfonyl fluoride, 1 μg/mL aprotinin). Sample aliquots wereresolved by SDS polyacrylamide gel electrophoresis (PAGE) on 15%acrylamide/0.5% bis-acrylamide gels and electrophoretically transferredto nitrocellulose. Non-specific binding was blocked by preincubatingblots in Blotto (phosphate buffered saline containing 5% w/v non-fatdried milk and 0.2% v/v nonidet P-40 (Sigma)), and recombinant proteinwas detected using a murine Mab to the HA decapeptide tag.Alternatively, recombinant protein can be detected using various AUR1-or AUR2-specific antisera.

[0240] Results:

[0241] Recombinant AUR1 and AUR2 expressed in COS cells migrated withapparent Mr of 39,000 and 46,000, consistent with their predictedmolecular weights of 39264 and 46730 based on their primary amino acidsequence. This analysis confirms that the recombinant protein can bestably produced in mammalian cells.

[0242] Dominant negative and constitutively active forms of AUR1 andAUR2 will be useful for delineating biologic consequences of eitheroblation or overexpression of these putative serine/threonine kinases.Initial studies with altered DNA constructs demonstrates that in just 2hours following infection of NIH3T3 or BALB/3T3 cells with aur1or aur2retroviral stocks, cells become multinucleated. This phenotype persistedsuch that 2 days after infection some cells were found to have as manyas 20 nuclei. The multinucleated cells typically had increased cytoplasmand diffuse cell boundaries. Immunostaining with both actin and DAPI,confirmed these nuclei were all contained within a single cell, and thatthe actin cytoskeleton was apparently normal.

Example 4 Generation of AUR-Specific Immunoreagents

[0243] Material and Methods:

[0244] AUR-specific immunoreagents were raised in rabbits againstKLH-conjugated synthetic peptides corresponding to either the N-terminalregion of AUR2 (¹⁰⁴SAPENNPEEQLASK¹¹⁷) (SEQ ID NO:22) and(⁹⁰RPLNNTQKSKQPL¹⁰²) (SEQ ID NO:23) or the N-terminus or N-terminaldomain of human AUR1 (¹MAQKENSYPWPYG¹³) (SEQ ID NO:24) and(⁵³PGQKVMENSSGTP⁶⁵) (SEQ ID NO:25). Additional immunoreagents weregenerated by immunizing rabbits with the bacterially expressed fulllength AUR1 and AUR2 GST-fusion proteins.

[0245] Results:

[0246] Specific immunoreagents were generated in rabbits against peptidesequence from the N-terminal domains of AUR1 and AUR2 to localizeexpression of endogenous and recombinant AUR within cells. Thesereagents can also be used to identify substrates for the AURs.

Example 5 Myelin Basic Protein is an Artificial Substrate for AUR1 andAUR2 Kinase

[0247] Method:

[0248] Human colorectal adenocarcinoma SW480 cells were cultured in RPMI1640 plus 10% fetal bovine serum, L-glutamine, penicillin andstreptomycin. Confluent cultures of SW480 cells were washed three timeswith ice cold phosphate buffered saline (PBS) and then were scraped into1 mL of ice cold PBS. The cells were centrifuged at 1,000 rpm at 4° C.,the PBS aspirated away, and the resulting cell pellet stored at −80° C.The pellets from three 15 cm plates were thawed on ice and resuspendedin a total of 1 mL of kinase lysis buffer 50 mM HEPES pH 7.4, 100 mMKCl, 25 mM NaF, 1 mM NaVO₃, 0.5% NP40, 1 mM DTT, 2 μg/mL aprotinin, and1 μg/mL leupeptin) and were rotated gently for 20 minutes at 4° C. Thesamples were then centrifuged at 10,000×g for 10 minutes at 4° C. andthe resulting supernatant was transferred to a clean 1.5 mL centrifugetube and stored or kept on ice. The protein concentration was determinedby Bradford analysis. One milligram of total protein was pre-clearedwith 10 μL of protein A-Sepharose (Boehringer) for 15 minutes at 4° C.followed by the addition of 2 μL of either rabbit pre-immune serum,affinity purified AUR1 peptide antisera, affinity purified AUR1 peptideantisera plus 6 μg of competing AUR1 peptide, affinity purified AUR2peptide antisera, or, affinity purified AUR2 peptide antisera plus 6 μgof competing AUR2 peptide and incubated for 30 minutes at 4° C.Subsequently, 10 μL of protein A-sepharose was added and the incubationwas continued for an additional 45 minutes at 4° C. The tubes werebriefly centrifuged to pellet the antibody-protein A-sepharose complexand the resulting supernatant was aspirated off. The antibody-proteinA-sepharose pellet was washed twice with 0.5 mL of kinase lysis bufferfollowed by a wash with 0.5 mL of kinase buffer (20 mM HEPES pH 7.4, 125mM KCl, 10 mM MgCl₂, 1 mM NaF, 1 mM NaVO₃, and 1 mM DTT). Theantibody-protein A-sepharose pellet was resuspended in 20 μL of kinasebuffer containing 5 μCi of [γ-³²P] ATP and 0.5 mg/mL myelin basicprotein (Sigma), incubated for 20 minutes at 37° C. after which 10 μL ofprotein sample buffer 200 mM Tris-Cl pH 6.8, 40% glycerol, 730 mMB-mercaptoethanol, 0.4% SDS, and 0.05% Bromophenol Blue) was added. Thetubes were mixed well and incubated for 5 minutes at 100° C. The sampleswere resolved on an 18% SDS polyacrylamide gel and visualized byautoradiography.

[0249] Results:

[0250] AUR1 and AUR2 immunocomplexes were able to phosphorylate myelinbasic protein. When competing peptide was used in theimmunoprecipitations neither AUR1 nor AUR2 antsera immunocomplexes wereable to phosphorylate myelin basic protein more than the pre-immune seracontrol. This suggests that the kinase activity observed is due to AUR1and AUR2 and not to other proteins present in the immunocomplex.

[0251] This observation will allow for the purification of active AUR1and AUR2 kinase by using myelin basic protein as a substrate to followkinase activity. It also will allow the development of an in vitrokinase assay using recombinant AUR1 and AUR2 proteins. Furthermore anAUR1 and AUR2 in vitro kinase assay will allow one to screen smallmolecule collections for inhibitors of the AUR1 and AUR2 kinases bymeasuring the inhibition of phosphorylation of myelin basic protein.

Example 6 Structural Comparison of Aur Homologues

[0252] Materials and Methods:

[0253] cDNA cloning

[0254] Degenerate oligonucleotide primers were designed for PCR cloningbased on kinase domains I and IX of CCK4 (GenBank:U33635; Cowley et al.,Cell 77:841-852, 1994), a receptor tyrosine kinase expressed in a widerange of normal and transformed epithelial cells. The sense primer was5′-GARTTYGGNGARGTNTTYYTNGC-3′ (SEQ ID NO:16), encoding the amino acidsEFGEVFLA (SEQ ID NO:18) and the antisense primer was5′-AGNACNCCRAANGCCCACACRTC-3′ (SEQ ID NO:17), encoding the complementarystrand of amino acids DVWAFGVL (SEQ ID NO:33). These primers wereapplied to sscDNA generated from RNA isolated from several colon cancercell lines as well as other tumor sources. PCR products of 500-600 bpwere subcloned and sequenced, revealing a fragment related to Drosophilaaurora. This fragment was used to probe a lambda library constructedfrom a pool of several human pancreatic cancer cell line RNAS, leadingto isolation of full length clones for human aur1. Two weaklyhybridizing clones were also isolated and sequence analysis revealedthat they represented a related but distinct cDNA termed aur2. Fulllength clones were also isolated for both genes from normal humanduodenum cDNA. All clones were sequenced on both strands with internaloligonucleotide primers using both T7 polymerase manual sequencing andusing dye-terminator cycle sequencing with AmpliTaq DNA polymerase on anABI Prism 377. The complete aur2 coding sequence was also confirmed from10 primary colorectal tumor samples. Primers 5′-CGCCTTTGCATCCGCTCCTG-3′(SEQ ID NO:34) and 5′-GATTTGCCTCCTGTGAAGAC-3′ (SEQ ID NO:35) were usedin an RT-PCR reaction with sscDNA generated from the tumor RNAs. The PCRproducts were purified by GeneClean and sequenced directly bydye-terminator cycle sequencing with several oligonucleotide primers.While no sequence differences were observed between clones isolated fromnormal or tumor sources, a single nucleotide polymorphism was identifiedin 2 of the tumor samples which would encode an F to I change at residue31. Abbreviations for degenerate nucleotide residues are: R=A or G; Y=Cor T; N=A, C, G or T.

[0255] Results:

[0256] A PCR-based screen was initiated to identify novel colon cancerassociated kinases. One of these clones encoded a protein with homologyto the aurora protein kinase from Drosophila melanogaster and the IPL1kinase from Saccharomyces cerevisiae (Francisco et al., Mol. Cell. Biol.14:4731-4740, 1994; Glover et al., Cell 81:95-105, 1995). While usingthis fragment to screeen for a full length cDNA clone, a weaklyhybridizing clone was found to encode a related kinase. These genes arereferred to as aur1 and aur2, to reflect their homology to each otherand to the Drosophila aurora kinase.

[0257] Aur1 cDNA contained a 1032 bp open reading frame that encodes a344 amino acid polypeptide with a predicted molecular mass of 39.3 kDa.The aur2 cDNA contained a 1209 bp open reading frame that encodes a 403amino acid polypeptide with a predicted molecular mass of 45.8 kDa. Twoadditional human aur pseudogenes were also identified as expressedtranscripts that are each contained on single exons and maintainstriking DNA homology to either aur1 or aur2, yet exhibit multiple frameshifts.

[0258] A partial sequence of BTAK (Sen et al., Oncogene 14:2195-2200,1997), a breast tumor associated kinase, has been reported that appearsto be fragment of human aur2. A second manuscript reports the sequenceof human aik (Kimura et al., J. Biol. Chem. 272:13766-13771, 1997), acell cycle-regulated protein localized to spindle pole bodies, whichshares 92% amino acid sequence identity with human AUR2 but is likelyidentical except for the incorporation of 6 frameshifts resulting fromsequencing errors. A third paper provides the sequence of AYK1 (Yanai etal. Oncogene 14:2943-2950, 1997), a meiotic regulated gene, that appearsto be the murine orthologue of AUR2. The present invention describes thefirst complete sequence for both human aur1 and aur2.

[0259] The deduced amino acid sequences of human aur1 and aur2 arepresented in FIG. 1 aligned with the yeast and Drosophila homologuesIPL1 and aurora. Human AUR2 protein shares 57%, 43%, and 41% identityover its entire length with human AUR1, Drosophila aurora, and IPL1,respectively. The four sequences contain a C-terminal domain with allthe characteristic motifs of a serine/threonine kinase. The kinasedomain of human AUR2 shares 74%, 62%, and 49% amino acid identity withhuman AUR1, Drosophila aurora, and IPL1 and 83.5% identity with twoamphibian homologues present in Xenopus [p46Eg22 (PIR:S53342) andp46Eg265 (PIR:S53343)]. The Drosophila aurora is most related to humanAUR1 whereas the yeast IPL1 is most related to AUR2. This structuralassessment is supported by complementation studies in yeast where onlythe human AUR2 kinase can complement an IPL1 mutant. Whereas a singleaurora-like kinase is present in yeast, at least two members are presentin C. elegans (GB:U53336, gene K07C11.2 and GB:U97196, gene B0207.4 Thededuced catalytic domains of these C. elegans proteins share 55% and 64%amino acid sequence identity to the human AUR2 kinase domain. We predictan additional aurora homologue will ultimately be identified inDrosophila as characterization of its genome nears completion.

[0260] The 129 and 73 amino acid N-terminal domains of human AUR2 andAUR1 share limited homology with each other and with the analogous 160and 100 amino acid domains of Drosophila aurora and yeast IPL1. TheN-terminal regions of human and mouse AUR2 share 54% identity to eachother and 28-30% identity to the two Xenopus proteins, and together helpdefine two distantly conserved motifs present in the non-catalyticregion of all auroras (FIG. 1). The first motif includes 10 aminostretch, KENX₄PVK, termed AUR Box1 and the second motif is centeredaround a 15 amino acid stretch, QX₉AQRVL, termed AUR Box 2 (seeoverlines in FIG. 1). The Drosophila aurora has a 36 amino acid insertin AUR Box2. Several potential serine and threonine phosphorylationsites are also conserved among these proteins including a protein kinaseA phosphorylation motif RRXT in the activation loop of the kinase. Atemperature sensitive mutant of the yeast IPL1 gene consists of a Thr toAla substitution within the activation loop (Gopalan et al., J. CellBiol. 138:643-656, 1997), suggesting that phosphorylation at this sitemay be biologically relevant. Additional mutants in the yeast (Chan1993, supra) and Drosophila (Glover 1995, supra) homologues of aurorahave been mapped exclusively to the kinase domain, except for a singleDrosophila mutant (Glover 1995, supra) that involves a mutation at Asp47within the N-terminal AUR Box2. Since these mutations result in abnormalnuclei, chromosome missegregation, and monopolar spindles, thesefindings suggest that the catalytic activity of the auroras may play animportant role in centrosome biology.

Example 7 Expression of Aur1 and Aur2 RNA in Normal Tissues and TumorCell Lines

[0261] Materials and Methods:

[0262] Northern Blots

[0263] Cell pellets from cultured tumor cell lines were provided by NickScuidero (Developmental Therapeutics Program, NCI), and are part of theNCI tumor panel (see website listing athttp://epnws1.ncifcrf.gov:2345/dis3d/cancer_screen/celllist.html).Normal human tissue samples were obtained from the Cooperative HumanTissue Network (Cleveland, Ohio). Human colorectal tissue samples forNorthern and Southern analysis were obtained from Los Angeles areahospitals including UCLA-Harbor, Wadsworth and Cedars Sinai from 1988 to1997. Tumor histology was confirmed prior to preparing RNA, DNA andprotein lysates from each sample. Total cell or tissue RNA was isolatedusing the guanidine salts/phenol extraction protocol of Chomczynski andSacchi (Wolf et al., Oncogene 14:543-549, 1997). Northern blotting wasperformed using standard techniques (Mossie et al. Oncogene11:2179-2184, 1995) with a random-labeled 586 bp BamHI-SspI fragment ofthe human aur2 cDNA. A multiple tissue Northern blot and a human immunesystem blot (Clontech) containing 2 μg polyA mRNA per lane were alsoprobed for aur2 expression. A human b-actincDNA probe (Clontech) wasused to confirm equivalent loading of intact RNA. RNA (10 μg) from theSK-HEP-1 (HTB52) liver adenocarcinoma cell line served as an internalstandard for detection of aur2 expression on each blot. Blots werequantitated using a phosphorimager and ImageQuant software (MolecularDynamics, Mountain View, Calif.).

[0264] Results:

[0265] Northern blot analysis of mRNA isolated from normal adult humantissues demonstrates that aur2 expression is primarily restricted totestis, thymus and fetal liver, with very weak expression in bonemarrow, lymph node, and spleen and no detectable expression in all otheradult tissues examined. Additional studies demonstrate tight temporalregulation of these transcripts during mitosis (and Kimura 1997, supra).Human aur1 was also expressed at highest levels in normal testis andthymus, with a moderate level of expression in lung and small intestine.

[0266] Since these genes were originally identified from human tumors,we performed Northern blot analysis with aur2 on a panel of human tumorcell lines of colon, renal, melanoma, and breast origin. The 2.4 kb aur2transcript was expressed in 96% (24 of 25) of these transformed celllines, with the only exception being the UO-31 renal carcinoma cellline. The 1.4 kb aur1 transcript was co-expressed at surprisinglysimilar levels as aur2 in the same 24 tumor cell lines.

Example 8 Amplification and Overexpression of Aur2 in Primary HumanColorectal Cancers

[0267] Materials and Methods:

[0268] Chromosomal Localization

[0269] The Stanford G3 radiation hybrid panel was obtained from ResearchGenetics (Huntsville, Ala.). Primers used for radiation hybrid mappingwere: 5′-ATGCCTCCGGAAAGAGCCTGT-3′ (SEQ ID NO:36) and5′-GTGTCCCACTGCTATTCTCCAT-3′ (SEQ ID NO:37) for aur1 and5′-CAGGGCTGCCATATAACCTGA-3′ (SEQ ID NO:38) and5′-CTAGCACAGGCTGACGGGGC-3′ (SEQ ID NO:39) for aur2. The aur1 primers aredirected to the N-terminal region of the gene and generate a 247 bpfragment following 25 cycle PCR with a 54° C. annealing temperature. Theaur2 primers amplify a 255 bp fragment from the 3′ UTR following a 25cycle PCR with a 54° C. annealing temperature. The raw score for aur1against the SHGCR G3 panel is:00000000000010000000000000000000000000010000000100100100000000000100000000001010101, and for aur2:10000000001000101000000100010010000000000010000001100001001001010000010010010010010.

[0270] Southern Blotting

[0271] Genomic DNA was isolated from the human colorectal tissue samplesby standard methods (Proteinase K digestion, phenol:chloroformextraction, and ethanol precipitation). Southern blots were prepared bydigesting 5 μg of DNA with PstI, separating the fragments on 1% agarosegels, blotting onto nylon membranes (Nytran-Plus, Schleicher & Schuell),and probing sequentially with a random primer labeled 1044 bp aur2 cDNAfragment (pSG19) and a 1700 bp cloned fragment of the CYP24 gene(pKS-h24, from J. Omdahl, U. of New Mexico). A probe for human β-globinwas used to confirm equivalent sample loading. Final washes were at0.1×SSC, 0.1% SDS, 60° C. Autoradiographs were quantitated relative toβ-globin using ImageQuant software (Molecular Dynamics, Mountain View,Calif.).

[0272] Results:

[0273] Aur2 expression was next characterized by Northern blot analysisin a panel of 41 primary human colorectal tumors and matched normalcolorectal tissue from the same patients. Approximately 54% (22/41) ofthe samples showed increased expression of the 2.4 kb aur2 transcript inthe tumor as compared to the normal colon control. Aur2 RNA showed 4-28fold overexpression in tumor versus normal tissue.

[0274] The aur1 and aur2 genes were mapped using the Stanford HumanGenome Center G3 radiation hybrid panel. Human aur1is located onchromosome 17p13.1 (LOD score of 9.555 to linked marker SHGC-35513) andhuman aur2 on chromosome 20q13.2 (LOD score of 17.26 to linked markerSHGC-3245). Mapping was also confirmed by hybridization to ahuman-rodent somatic cell hybrid panel (Coriell Cell Repository, Camden,N.J.). Aur2 maps adjacent to the vitamin D hydroxylase (CYP24) gene andthe cosmid probe RMC20C001 that lie at 0.825-0.83 Flpter (fractionallength from pter) on chromosome 20 (Tanner 1994, supra; Tanner 1996,supra). Both of these markers have been characterized for their presencein the 20q13 amplicon common to many human malignancies, particularlythose from breast, bladder, and colon cancers.

[0275] Southern blot hybridization was performed using an aur2 cDNAprobe along with a control probe for the CYP24 gene that serves as amarker of the amplicon (Tanner 1994, supra; Tanner 1996, supra). Theaur2 probe hybridized to PstI fragments of 5.8, 3.7, 3.3, 2.8, 2.5, and1.3 kb. The 5.8, 3.3, 2.8, and 2.5 kb bands are specific to aur2. Onlythe aur2-specific bands showed amplification in the tumor samples.

[0276] Aur2 DNA was amplified in 41 of 79 (52%) of the primarycolorectal tumors for which suitable DNA was available for genotyping.Nine of twelve samples demonstrated a 2-8 fold amplification of aur2 DNAin the tumors compared to normal tissue. One of the samples demonstratesRNA overexpression in the absence of DNA amplification, whereas theother eleven show a direct correlation between DNA amplification and RNAoverexpression. The CYP24 gene was found to be co-amplified with aur2 in37 of 41 (90%) matched pairs, and was only once found to be amplified inthe absence of aur2 amplification.

[0277] There is a high correlation (p=0.695) between aur2 DNAamplification and RNA overexpression with only one discordant result. Inthe single case of aur2 DNA amplification in the absence of RNAoverexpression, aur2 RNA was actually elevated in both the normal andtumor specimens, compared to other tumor-normal pairs. Conceivably, highexpression of aur2 RNA in this normal colon sample may represent anearly predisposing lesion. Conversely, five paired samples showedincreased RNA expression in the absence of DNA amplification, possiblydue to transcriptional activation. If these five pairs are excluded fromthe analysis, the correlation between aur2 DNA amplification and RNAoverexpression increases to ρ=0.939. These data suggest that DNAamplification is a mechanism for AUR2 activation and also implicatesaur2 as an oncogene at 20q13 whose high level amplification correlateswith poor clinical outcome in breast cancer (Isola 1995, supra).

[0278] To determine if the aur2 sequence from the 20q13 amplicon was thesame as that from normal sources, we performed direct sequencing ofRT-PCR products encompassing the complete aur2 coding region from 10primary colorectal tumor samples. Eight samples, including both normaland amplified levels of the 20q13 amplicon, confirmed the aur2 sequence.A single nucleotide polymorphism was identified in 2 samples resultingin a phenylalanine to isoleucine change at residue 31 in the N-terminalAurora Box1 (circled in FIG. 1). This analysis demonstrates that the20q13 amplicon typically contains increased copies of the intact,unmutated aur2 coding region.

Example 9 Detection of AUR2 Protein in Primary Human Colon CancerSamples

[0279] Materials and Methods:

[0280] Western Blotting

[0281] Matched human tissue samples from primary colorectal carcinomasand adjacent normal tissue were obtained from the Cooperative HumanTissue Network (Cleveland, Ohio) and Pathology Associates International(Frederick, Md.). Thirty micron cryostat sections of OCT embedded tissuewas lysed directly in 25 μL of ice-cold RIPA buffer (50 mM Tris-Cl, pH8.0, 150 mM NaCl, 1.0% NP-40, 0.5% deoxycholate, 0.1% SDS, 1 mM DTT, andprotease inhibitors) by gentle mixing on ice for 20 minutes. The lysatewas then spun for 10 minutes at 10,000 ×g in a microfuge at 4° C. Theresulting supernatant was transferred to a clean tube and the totalprotein concentration was determined by Bradford analysis. Equal amountsof total protein from the matched samples was resolved on a 12%polyacrylamide gel, transferred to a nylon membrane (BioRad), and probedwith a 1:2,000 dilution of affinity purified antibodies to AUR2. Theimmunoblot was developed with ECL reagent (Amersham). Lysates from tumorcell lines were prepared and analysed as described above.

[0282] Results:

[0283] To analyze AUR2 protein expression, we generated polyclonalantibodies in rabbits against the entire open reading frame expressed inE. coli as a GST-fusion protein. The anti-AUR2 antibodies were used toprobe blots of protein lysates made from cryostat sections of primaryhuman colon carcinomas or from adjacent normal tissue. The AUR2antibodies detect a protein of approximately 46 kDa in two primary humancolon carcinomas, but not in samples derived from the adjacent normaltissue. These antibodies also detect overexpression of AUR2 protein invarious cultured tumor cell lines derived from colorectal carcinomas.

Example 10 Aur2 Transforms Rat1 Fibroblasts

[0284] Materials and Methods:

[0285] Expression Constructs

[0286] HA-tagged⁴² versions of wild-type, kinase dead (K162M), andactivated (T288D) aur2 were subcloned into the expression vector pLXSN.These contructs were transfected into the amphotropic packaging cellline PA317 and the supernatants were harvested and used to infect theproducer cell line GP+E-86 (Markowitz et al, Virology 167:400-406,1988). Neomycin resistant clones were selected and assayed for AUR2protein expression. Supernatants from the positive producer cell lineswere used to infect Rat1 and NIH3T3 cells. Stable clones were selectedfor in the presence of neomycin and assayed for AUR2 protein expressionby immunoblotinG.

[0287] In vitro Kinase Assays

[0288] Rat1 and NIH3T3 cells expressing the appropriate aur2 constructwere solubilized in kinase lysis buffer (50 mM Hepes, pH 7.4, 100 mMKCl, 25 mM NaF, 0.5w NP-40, 1 mM NaVO₃, 1 mM DTT, and proteaseinhibitors) for 15 minutes on ice, spun in a microfuge at 10,000×g for10 minutes at 4° C. The resulting supernatant was transferred to a cleantube and the total protein concentration was determined by Bradfordanalysis. Equal amounts of protein (usually 2 mg) wereimmunoprecipitated with the anti-HA monoclonal antibody 12CA(Boehinger). The immune complexes were washed three times with kinaselysis buffer followed and were either resuspended in 1×Laemmli SDSsample buffer or washed three times with kinase buffer (withoutγ-³²P-ATP and α-casein) and resuspended in 30 μL of 1×kinase buffer (20mM Hepes, pH 7.4, 150 mM KCl, 5 mM MnCl₂, 5 mM NaF, 1 mM DTT, 50 μM ATP,20 μCi γ-³²P-ATP, and 0.5 mg/mL α-casein). In vitro kinase reactionswere carried out for 20 minutes at 37° C. and stopped by the addition of30 μL of 2×Laemmli SDS sample buffer. Samples were incubated for 5minutes at 95° C. and resolved on 14% SDS-polyacrylamide gels.

[0289] Soft Agar Assays

[0290] A 3% solution of agar (at 56° C.) was diluted to a finalconcentration of 0.6% with growth medium (at 56° C.), pipetted intotissue culture dishes, and allowed to solidify at room temperature for20-30 minutes. At this time, 2×10⁵ cells in a volume of 50 μL were mixedwith 0.3% agar (diluted with growth media at 40° C.), pipetted gentlyonto the bottom agar layer, and allowed to solidify for 20-25 minutes atroom temperature. Once solidified, the plates were incubated at 37° C.in a 5% CO₂ atmosphere. Fresh top agar was added once a week. After 4weeks the plates were stained with neutral red.

[0291] Results:

[0292] If aur2 is a relevant target on the 20q13 amplicon, one mightexpect that overexpression of aur2 would be transforming. To examinethis question, we established stable Rat1 cell lines that express humanaur2. Rat1 cells were infected with retroviruses that express ahemagglutinin (HA)-tagged (Pati 1992, supra) wild-type aur2 or a kinaseinactive mutant where the essential lysine at residue 162 (FIG. 1) waschanged to a methionine (K162M). In addition, an activating mutation wasmade in which the threonine at residue 288 (FIG. 1) in the activationloop was changed to an aspartic acid (T288D). This mutation was designedto mimic constitutive phosphorylation at this site and indeed activatesthe kinase in vitro. The stable Rat1 lines expressed similar amounts ofAUR2 protein. In vitro kinase assays were performed using the AUR2protein produced by the stable cell lines. The kinase inactive AUR2mutant (K162M) was unable to phosphorylate α-casein over the levelsobserved in the vector control cell line in vitro, whereas the wild-typeand activated AUR2 (T288D) proteins had increased activity on thisartificial substrate.

[0293] To characterize the transforming potential of aur2, we performedsoft agar assays with the Rat1 clones. The vector control, K162M,wild-type, and T288D AUR2 expressing Rat1 cells were plated in soft agarand scored for growth after 4 weeks. Cells expressing the wild-type andthe T288D AUR2 formed colonies in soft agar, in contrast to the lack ofgrowth by cells expressing the kinase inactive AUR2. Ten of thirteenwild-type clones and six of twelve T288D clones grew in soft agar,compared to one of eleven vector and K162M clones. The number ofcolonies formed in soft agar from two independent clones of each of thetransfections was quantitated. The average number of colonies per200,000 cells plated were: K162M, 32 colonies; wild-type, 470 colonies;and T288D, 250 colonies. Although the T288D Rat1 stables formed fewercolonies than the wild-type AUR2 Rat1 stables, the T288D colonies ingeneral grew to larger size.

[0294] The T288D AUR2 mutant was also able transform NIH3T3 cells, asmeasured by growth in soft agar and growth as tumors in nude mice. Inconstrast, the wild-type AUR2 was unable to transform NIH3T3 cells. Thetransforming ability of these constructs correlated with catalyticactivity, since the wild-type AUR2 was catalytically inactive in NIH3T3cells whereas the T288D had strong kinase activity. These data suggestthat the genetic background of the cells is important in determining thetransforming potential of AUR2.

Example 11 Activated AUR Transforms NIH3T3 Cells

[0295] Stable mouse NIH3T3 cell lines that express human AUR2 wereestablished. NIH3T3 cells were infected with retroviruses that express ahemagglutinin (HA)-tagged (Chan 1993, supra) wild-type AUR2 or a kinaseinactive mutant where the essential lysine at residue 162 (FIG. 1) waschanged to a methionine (K162M). In addition, an activating mutation wasmade in which the threonine at residue 288 (FIG. 1) in the activationloop was changed to an aspartic acid (T288D). This mutation was designedto mimic constitutive phosphorylation at this site and indeed activatesthe kinase in vitro. The stable lines expressed similar amounts of AUR2protein. In vitro kinase assays were performed using the AUR2 proteinproduced by the stable cell lines.

[0296] The wild-type and kinase inactive AUR2 mutant (K162M) were unableto phosphorylate myelin basic protein in vitro, however the activatedAUR2 mutant (T288D) had marked activity on this artificial substrate. Itis not clear why the wild-type AUR2 appears to be catalytically inactivein the in vitro kinase assay. Perhaps a putative activator of AUR2 islimiting in the NIH3T3 cells or a negative regulator, such as an AUR2phosphatase, has increased activity in these cells. Indeed, aninterfering mutant of the S. cerevisiae phosphatase PP1 can exacerbatethe IPL1 mutant phenotype, suggesting that IPL1 may be negativelyregulated by this phosphatase (Francisco et al, Mol. Cell. Biol.14:4731-4740). It is also possible that while activated AUR2 can utilizemyelin basic protein as an artificial substrate in vitro, the wild-typekinase has a more restricted substrate preference.

[0297] To determine if ectopic expression of activated AUR2 alters thegrowth of NIH3T3 cells, growth curves in media containing low (2%) serumwere generated. For the first 24 hours under these conditions, theexpression of activated AUR2 provided a growth advantage to NIH3T3 ascompared to cell lines expressing wild-type AUR2, kinase inactive AUR2,or the vector control. However, after 48 hours in 2% serum, all the celllines ceased to divide, indicating that activated AUR2 alone is unableto promote indefinite cell proliferation.

[0298] To characterize the transforming potential of activated AUR2,soft agar assays were performed with the 3T3 clones. The vector control,wild-type, kinase inactive, and activated AUR2 expressing NIH3T3 cellswere plated in soft agar and scored for growth after 3-4 weeks. Cellsexpressing the activated AUR2 grew large colonies in soft agar, in sharpcontrast to the lack of growth by cells expressing the wild-type and thekinase inactive AUR2. Ectopic expression of activated AUR2 appears toconfer a growth advantage to NIH3T3 cells in low serum and results inanchorage-dependent growth.

Example 12 Aur2 Antisense Oligos Inhibit AUR2 Expression In Vivo

[0299] Material and Methods:

[0300] Human H1299 cells were seeded at ˜40-50% confluency in a 6 wellplate (Falcon). The following day lipofectin (Gibco) and oligo(s) weremixed with OptiMEM (Gibco) such that the final concentration oflipofectin is 100 μg/mL and the final concentration of each oligo is 1μM in a volume of 200 μL. The lipofectin/oligo/OptiMEM mixture wasincubated at room temperature (20-25° C.) for 15 minutes, 800 μL ofOptiMEM was added to the lipofectin/oligo/OptiMEM mixture, and mixedgently. The growth medium was removed from the H1299 cells, which wereat ˜80% confluency. Cells were washed once with OptiMEM. The OptiMEM wasaspirated, the lipofectin/oligo/OptiMEM mixture was added, culturesincubated at 37° C. for 4 hours. The lipofectin/oligo/OptiMEM mixturewas removed and replaced with normal growth medium containing theantisense oligo(s) at a concentration of 200 nM. The plates werereturned to the 37° C. incubator for 16-20 hours after which the growthmedium was removed from the cells and they were washed once withOptiMEM. The OptiMEM was again aspirated and thelipofectin/oligo/OptiMEM mixture was added (prepared as described above)and the plates again were incubated at 37° C. for 4 hours. Thelipofectin/oligo/OptiMEM mixture was removed again and replaced withnormal growth medium containing the antisense oligo(s) at aconcentration of 200 nM. The platea were returned to the 37° C.incubator for 16-20 hours. The cells were harvested (˜40 hours afterinitial treatment) and analyzed for aur2 mRNA by northern blot or AUR2protein expression by immunoblot.

[0301] Results:

[0302] Three antisense oligonucleotides (SEQ ID NOs:16, 17, and 18)which target specific regions of human aur2 mRNA transcript were foundto significantly down regulate AUR2 protein expression in the humantumor cell line H1299. When used in combination oligos SEQ ID NO:30 andSEQ ID NO:32 and SEQ ID NO:31 and SEQ ID NO:32 reduce the expression ofAUR2 protein below the limit of detection. Treatment of H1299 cells withthe combination of SEQ ID NO:31 and SEQ ID NO:32 inhibited the growth ofthis tumor cell line as measured by cell growth and appeared to induceapoptosis as measured by FACs.

[0303] Sequences (5′-3′): The oligo number as well as the location ofthe sequence within the aur2 cDNA are presented below. Note: theseoligonucleotides were synthesized as phosphothionates. SEQ ID NO:30(nucleotides 1743-1763): CAGGGCAGAGTGGTCACTTTC SEQ ID NO:31 (nucleotides42-62): CGTCCGCCACTCCGACCAGCC SEQ ID NO:32 (nucleotides 1654-1674):TGCAGTCGAACCTTGCCTCCA

[0304] These antisense oligonucleotides would be useful for inhibitionof AUR2 expression in normal and tumor cells in order to profile thepotential effects of small molecule AUR2 inhibitors. They can also beused to inhibit AUR2 expression in human tumor cell xenografts in nudemice to determine the antitumor effects of AUR2 inhibitors. Another useis as a “drug” to inhibit AUR2 expression in various human tumors thatare “driven” by overexpression of the aur2 gene.

Example 13 Screening Systems for the Identification of Inhibitors ofAUR2 Activity

[0305] Assays may be performed in vitro or in vivo and are described indetail herein or can be obtained by modifying ” existing assays, such asthe growth assay described in patent application Ser. No. 08/487,088(Lyon & Lyon Docket No. 212/276), filed Jun. 7, 1995, by Tang et al.,and entitled “Novel Pharmaceutical Compounds”, or the assays describedin patent application Ser. No. 60/005,167 (Lyon & Lyon Docket No.215/256), filed Oct. 13, 1995 by Seedorf et al., and entitled “Diagnosisand Treatment of TKA-1 related disorders”, all of which are herebyincorporated herein by reference in their entirety including anydrawings. Another assay which could be modified to use the genes of thepresent invention is described in International Application No. WO94/23039, published Oct. 13, 1994, hereby incorporated herein byreference in its entirety including any drawings. Other possibilitiesinclude detecting kinase activity in an autophosphorylation assay ortesting for kinase activity on standard substrates such as histones,myelin basic protein, gamma tubulin, or centrosomal proteins. Bindingpartners may be identified by putting the N-terminal portion of theprotein into a two-hybrid screen or detecting phosphotyrosine of a dualspecificity kinase (Fields and Song, U.S. Pat. No. 5,283,173, issuedFeb. 1, 1994, incorporated by reference herein, including any drawings).

[0306] One means by which inhibitors of AUR2 activity may be defined isa screening system using a temperature-sensitive yeast mutant asdescribed by Chan and Botstein (Genetics 135:677-691, 1993); see alsoFrancisco et al. (Mol. Cell. Bio. 14:4731-4740, 1994) both of which arehereby incorporated herein by reference in their entirety including anydrawings.

[0307] Briefly, yeast strain CCY72-3D-1 (ipl 1-2), which expresses atemperature sensitive form of the yeast homologue of AUR2 (ip11), whileviable at 26° C. is incapable of growth at 37° C.. Transfection of thisstrain with an expression plasmid containing a hybrid aurora geneconsisting of the N-terminal portion of ip11, containing the putativesubstrate interaction domain(s)), and the C-terminal portion of AUR2,containing the catalytic domain, overcomes this sensitivity to growthtemperature. The AUR-expressing yeast strain is then grown at 37° C. inthe presence of a test substance. No growth will be evident in thepresence of substances that inhibit AUR catalytic function. Potentialinhibitors include antisense oligonucleotides, small molecular weightchemicals, and/or natural products isolated from diverse organisms suchas fungi, marine organisms, plants, etc.

[0308] One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Themolecular complexes and the methods, procedures, treatments, molecules,specific compounds described herein are presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art which are encompassed within the spirit ofthe invention are defined by the scope of the claims.

[0309] It will be readily apparent to one skilled in the art thatvarying substitutions and modifications may be made to the inventiondisclosed herein without departing from the scope and spirit of theinvention.

[0310] All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

[0311] The invention illustratively described herein suitably may bepracticed in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein. Thus, forexample, in each instance herein any of the terms “comprising”,“consisting essentially of” and “consisting of” may be replaced witheither of the other two terms. The terms and expressions which have beenemployed are used as terms of description and not of limitation, andthere is no intention that in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed.

[0312] In addition, where features or aspects of the invention aredescribed in terms of Markush groups, those skilled in the art willrecognize that the invention is also thereby described in terms of anyindividual member or subgroup of members of the Markush group. Forexample, if X is described as selected from the group consisting ofbromine, chlorine, and iodine, claims for X being bromine and claims forX being bromine and chlorine are fully described.

[0313] In view of the degeneracy of the genetic code, other combinationsof nucleic acids also encode the claimed peptides and proteins of theinvention. For example, all four nucleic acid sequences GCT, GCC, GCA,and GCG encode the amino acide alanine. Therefore, if for an amino acidthere exists an average of three codons, a polypeptide of 100 aminoacids in length will, on average, be encoded by 3¹⁰⁰, or 5×10⁴⁷, nucleicacid sequences. It is understood by those skilled in the art that, with,Thus, a nucleic acid sequence can be modified to form a second nucleicacid sequence, encoding the same polypeptide as endoded by the firstsecond nucleic acid sequences, using routine procedures and withoutundue experimentation. Thus, all possible nucleic acids that encode theclaimed peptides and proteins are also fully described herein, as if allwere written out in full taking into account the codon usage, especiallythat preferred in humans.

[0314] Furthermore, changes in the amino acid sequences of polypeptides,or in the corresponding nucleic acid sequence encoding such polypeptide,may be designed or selected to take place in an area of the sequencewhere the significant activity of the polypeptide remains unchanged. Forexample, an amino acid change may take place within a β-turn, away fromthe active site of the polypeptide. Also changes such as deletions (e.g.removal of a segment of the polypeptide, or in the corresponding nucleicacid sequence encoding such polypeptide, which does not affect theactive site) and additions (e.g. addition of more peptides to thepolypeptide sequence without affecting the function of the active site,such as the formation of GST-fusion proteins, or additions in thecorresponding nucleic acid sequence encoding such polypeptide withoutaffecting the function of the active site) are also within the scope ofthe present invention. Such changes to the polypeptides can be performedby those with ordinary skill in the art using routine procedures andwithout undue experimentation. Thus, all possible nucleic and/or aminoacid sequences that can readily be determined not to affect asignificant activity of the peptide or protein of the invention are alsofully described herein.

[0315] Other embodiments are within the following claims.

[0316] Other embodiments are within the following claims.

1 39 1244 base pairs nucleic acid single linear cDNA NO NO Homo sapiens1 CGGGAGAGTA GCAGTGCCTT GGACCCCAGC TCTCCTCCCC CTTTCTCTCT AAGGATGGCC 60CAGAAGGAGA ACTCCTACCC CTGGCCCTAC GGCCGACAGA CGGCTCCATC TGGCCTGAGC 120ACCCTGCCCC AGCGAGTCCT CCGGAAAGAG CCTGTCACCC CATCTGCACT TGTCCTCATG 180AGCCGCTCCA ATGTCCAGCC CACAGCTGCC CCTGGCCAGA AGGTGATGGA GAATAGCAGT 240GGGACACCCG ACATCTTAAC GCGGCACTTC ACAATTGATG ACTTTGAGAT TGGGCGTCCT 300CTGGGCAAAG GCAAGTTTGG AAACGTGTAC TTGGCTCGGG AGAAGAAAAG CCATTTCATC 360GTGGCGCTCA AGGTCCTCTT CAAGTCCCAG ATAGAGAAGG AGGGCGTGGA GCATCAGCTG 420CGCAGAGAGA TCGAAATCCA GGCCCACCTG CACCATCCCA ACATCCTGCG TCTCTACAAC 480TATTTTTATG ACCGGAGGAG GATCTACTTG ATTCTAGAGT ATGCCCCCCG CGGGGAGCTC 540TACAAGGAGC TGCAGAAGAG CTGCACATTT GACGAGCAGC GAACAGCCAC GATCATGGAG 600GAGTTGGCAG ATGCTCTAAT GTACTGCCAT GGGAAGAAGG TGATTCACAG AGACATAAAG 660CCAGAAAATC TGCTCTTAGG GCTCAAGGGA GAGCTGAAGA TTGCTGACTT CGGCTGGTCT 720GTGCATGCGC CCTCCCTGAG GAGGAAGACA ATGTGTGGCA CCCTGGACTA CCTGCCCCCA 780GAGATGATTG AGGGGCGCAT GCACAATGAG AAGGTGGATC TGTGGTGCAT TGGAGTGCTT 840TGCTATGAGC TGCTGGTGGG GAACCCACCC TTCGAGAGTG CATCACACAA CGAGACCTAT 900CGCCGCATCG TCAAGGTGGA CCTAAAGTTC CCCGCTTCTG TGCCCACGGG AGCCCAGGAC 960CTCATCTCCA AACTGCTCAG GCATAACCCC TCGGAACGGC TGCCCCTGGC CCAGGTCTCA 1020GCCCACCCTT GGGTCCGGGC CAACTCTCGG AGGGTGCTGC CTCCCTCTGC CCTTCAATCT 1080GTCGCCTGAT GGTCCCTGTC ATTCACTCGG GTGCGTGTGT TTGTATGTCT GTGTATGTAT 1140AGGGGAAAGA AGGGATCCCT AACTGTTCCC TTATCTGTTT TCTACCTCCT CCTTTGTTTA 1200ATAAAGGCTG AAGCTTTTTG TAAAAAAACA AAAAAAAAAA AAAA 1244 2198 base pairsnucleic acid single linear cDNA NO NO 2 GGGATATCTC AGTGGCGGAC GAGGACGGCGGGGACAAGGG GCGGCTGGTC GGAGTGGCGG 60 ACGTCAAGTC CCCTGTCGGT TCCTCCGTCCCTGAGTGTCC TTGGCGCTGC CTTGTGCCCG 120 CCCAGCGCCT TTGCATCCGC TCCTGGGCACCGAGGCGCCC TGTAGGATAC TGCTTGTTAC 180 TTATTACAGC TAGAGGCATC ATGGACCGATCTAAAGAAAA CTGCATTTCA GGACCTGTTA 240 AGGCTACAGC TCCAGTTGGA GGTCCAAAACGTGTTCTCGT GACTCAGCAA TTTCCTTGTC 300 AGAATCCATT ACCTGTAAAT AGTGGCCAGGCTCAGCGGGT CTTGTGTCCT TCAAATTCTT 360 CCCAGCGCGT TCCTTTGCAA GCACAAAAGCTTGTCTCCAG TCACAAGCCG GTTCAGAATC 420 AGAAGCAGAA GCAATTGCAG GCAACCAGTGTACCTCATCC TGTCTCCAGG CCACTGAATA 480 ACACCCAAAA GAGCAAGCAG CCCCTGCCATCGGCACCTGA AAATAATCCT GAGGAGGAAC 540 TGGCATCAAA ACAGAAAAAT GAAGAATCAAAAAAGAGGCA GTGGGCTTTG GAAGACTTTG 600 AAATTGGTCG CCCTCTGGGT AAAGGAAAGTTTGGTAATGT TTATTTGGCA AGAGAAAAGC 660 AAAGCAAGTT TATTCTGGCT CTTAAAGTGTTATTTAAAGC TCAGCTGGAG AAAGCCGGAG 720 TGGAGCATCA GCTCAGAAGA GAAGTAGAAATACAGTCCCA CCTTCGGCAT CCTAATATTC 780 TTAGACTGTA TGGTTATTTC CATGATGCTACCAGAGTCTA CCTAATTCTG GAATATGCAC 840 CACTTGGAAC AGTTTATAGA GAACTTCAGAAACTTTCAAA GTTTGATGAG CAGAGAACTG 900 CTACTTATAT AACAGAATTG GCAAATGCCCTGTCTTACTG TCATTCGAAG AGAGTTATTC 960 ATAGAGACAT TAAGCCAGAG AACTTACTTCTTGGATCAGC TGGAGAGCTT AAAATTGCAG 1020 ATTTTGGGTG GTCAGTACAT GCTCCATCTTCCAGGAGGAC CACTCTCTGT GGCACCCTGG 1080 ACTACCTGCC CCCTGAAATG ATTGAAGGTCGGATGCATGA TGAGAAGGTG GATCTCTGGA 1140 GCCTTGGAGT TCTTTGCTAT GAATTTTTAGTTGGGAAGCC TCCTTTTGAG GCAAACACAT 1200 ACCAAGAGAC CTACAAAAGA ATATCACGGGTTGAATTCAC ATTCCCTGAC TTTGTAACAG 1260 AGGGAGCCAG GGACCTCATT TCAAGACTGTTGAAGCATAA TCCCAGCCAG AGGCCAATGC 1320 TCAGAGAAGT ACTTGAACAC CCCTGGATCACAGCAAATTC ATCAAAACCA TCAAATTGCC 1380 AAAACAAAGA ATCAGCTAGC AAACAGTCTTAGGAATCGTG CAGGGGGAGA AATCCTTGAG 1440 CCAGGGCTGC CATATAACCT GACAGGAACATGCTACTGAA GTTTATTTTA CCATTGACTG 1500 CTGCCCTCAA TCTAGAACGC TACACAAGAAATATTTGTTT TACTCAGCAG GTGTGCCTTA 1560 ACCTCCCTAT TCAGAAAGCT CCACATCAATAAACATGACA CTCTGAAGTG AAAGTAGCCA 1620 CGAGAATTGT GCTACTTATA CTGGTTCATAATCTGGAGGC AAGGTTCGAC TGCAGCCGCC 1680 CCGTCAGCCT GTGCTAGGCA TGGTGTCTTCACAGGAGGCA AATCCAGAGC CTGGCTGTGG 1740 GGAAAGTGAC CACTCTGCCC TGACCCCGATCAGTTAAGGA GCTGTGCAAT AACCTTCCTA 1800 GTACCTGAGT GAGTGTGTAA CTTATTGGGTTGGCGAAGCC TGGTAAAGCT GTTGGAATGA 1860 GTATGTGATT CTTTTTAAGT ATGAAAATAAAGATATATGT ACAGACTTGT ATTTTTTCTC 1920 TGGTGGCATT CCTTTAGGAA TGCTGTGTGTCTGTCCGGCA CCCCGGTAGG CCTGATTGGG 1980 TTTCTAGTCC TCCTTAACCA CTTATCTCCCATATGAGAGT GTGAAAAATA GGAACACGTG 2040 CTCTACCTCC ATTTAGGGAT TTGCTTGGGATACAGAAGAG GCCATGTGTC TCAGAGCTGT 2100 TAAGGGCTTA TTTTTTTAAA ACATTGGAGTCATAGCATGT GTGTAAACTT TAAATATGCA 2160 AATAAATAAG TATCTATGTC AAAAAAAAAAAAAAAAAA 2198 344 amino acids amino acid single linear protein NO NO 3Met Ala Gln Lys Glu Asn Ser Tyr Pro Trp Pro Tyr Gly Arg Gln Thr 1 5 1015 Ala Pro Ser Gly Leu Ser Thr Leu Pro Gln Arg Val Leu Arg Lys Glu 20 2530 Pro Val Thr Pro Ser Ala Leu Val Leu Met Ser Arg Ser Asn Val Gln 35 4045 Pro Thr Ala Ala Pro Gly Gln Lys Val Met Glu Asn Ser Ser Gly Thr 50 5560 Pro Asp Ile Leu Thr Arg His Phe Thr Ile Asp Asp Phe Glu Ile Gly 65 7075 80 Arg Pro Leu Gly Lys Gly Lys Phe Gly Asn Val Tyr Leu Ala Arg Glu 8590 95 Lys Lys Ser His Phe Ile Val Ala Leu Lys Val Leu Phe Lys Ser Gln100 105 110 Ile Glu Lys Glu Gly Val Glu His Gln Leu Arg Arg Glu Ile GluIle 115 120 125 Gln Ala His Leu His His Pro Asn Ile Leu Arg Leu Tyr AsnTyr Phe 130 135 140 Tyr Asp Arg Arg Arg Ile Tyr Leu Ile Leu Glu Tyr AlaPro Arg Gly 145 150 155 160 Glu Leu Tyr Lys Glu Leu Gln Lys Ser Cys ThrPhe Asp Glu Gln Arg 165 170 175 Thr Ala Thr Ile Met Glu Glu Leu Ala AspAla Leu Met Tyr Cys His 180 185 190 Gly Lys Lys Val Ile His Arg Asp IleLys Pro Glu Asn Leu Leu Leu 195 200 205 Gly Leu Lys Gly Glu Leu Lys IleAla Asp Phe Gly Trp Ser Val His 210 215 220 Ala Pro Ser Leu Arg Arg LysThr Met Cys Gly Thr Leu Asp Tyr Leu 225 230 235 240 Pro Pro Glu Met IleGlu Gly Arg Met His Asn Glu Lys Val Asp Leu 245 250 255 Trp Cys Ile GlyVal Leu Cys Tyr Glu Leu Leu Val Gly Asn Pro Pro 260 265 270 Phe Glu SerAla Ser His Asn Glu Thr Tyr Arg Arg Ile Val Lys Val 275 280 285 Asp LeuLys Phe Pro Ala Ser Val Pro Thr Gly Ala Gln Asp Leu Ile 290 295 300 SerLys Leu Leu Arg His Asn Pro Ser Glu Arg Leu Pro Leu Ala Gln 305 310 315320 Val Ser Ala His Pro Trp Val Arg Ala Asn Ser Arg Arg Val Leu Pro 325330 335 Pro Ser Ala Leu Gln Ser Val Ala 340 403 amino acids amino acidsingle linear protein NO NO 4 Met Asp Arg Ser Lys Glu Asn Cys Ile SerGly Pro Val Lys Ala Thr 1 5 10 15 Ala Pro Val Gly Gly Pro Lys Arg ValLeu Val Thr Gln Gln Phe Pro 20 25 30 Cys Gln Asn Pro Leu Pro Val Asn SerGly Gln Ala Gln Arg Val Leu 35 40 45 Cys Pro Ser Asn Ser Ser Gln Arg ValPro Leu Gln Ala Gln Lys Leu 50 55 60 Val Ser Ser His Lys Pro Val Gln AsnGln Lys Gln Lys Gln Leu Gln 65 70 75 80 Ala Thr Ser Val Pro His Pro ValSer Arg Pro Leu Asn Asn Thr Gln 85 90 95 Lys Ser Lys Gln Pro Leu Pro SerAla Pro Glu Asn Asn Pro Glu Glu 100 105 110 Glu Leu Ala Ser Lys Gln LysAsn Glu Glu Ser Lys Lys Arg Gln Trp 115 120 125 Ala Leu Glu Asp Phe GluIle Gly Arg Pro Leu Gly Lys Gly Lys Phe 130 135 140 Gly Asn Val Tyr LeuAla Arg Glu Lys Gln Ser Lys Phe Ile Leu Ala 145 150 155 160 Leu Lys ValLeu Phe Lys Ala Gln Leu Glu Lys Ala Gly Val Glu His 165 170 175 Gln LeuArg Arg Glu Val Glu Ile Gln Ser His Leu Arg His Pro Asn 180 185 190 IleLeu Arg Leu Tyr Gly Tyr Phe His Asp Ala Thr Arg Val Tyr Leu 195 200 205Ile Leu Glu Tyr Ala Pro Leu Gly Thr Val Tyr Arg Glu Leu Gln Lys 210 215220 Leu Ser Lys Phe Asp Glu Gln Arg Thr Ala Thr Tyr Ile Thr Glu Leu 225230 235 240 Ala Asn Ala Leu Ser Tyr Cys His Ser Lys Arg Val Ile His ArgAsp 245 250 255 Ile Lys Pro Glu Asn Leu Leu Leu Gly Ser Ala Gly Glu LeuLys Ile 260 265 270 Ala Asp Phe Gly Trp Ser Val His Ala Pro Ser Ser ArgArg Thr Thr 275 280 285 Leu Cys Gly Thr Leu Asp Tyr Leu Pro Pro Glu MetIle Glu Gly Arg 290 295 300 Met His Asp Glu Lys Val Asp Leu Trp Ser LeuGly Val Leu Cys Tyr 305 310 315 320 Glu Phe Leu Val Gly Lys Pro Pro PheGlu Ala Asn Thr Tyr Gln Glu 325 330 335 Thr Tyr Lys Arg Ile Ser Arg ValGlu Phe Thr Phe Pro Asp Phe Val 340 345 350 Thr Glu Gly Ala Arg Asp LeuIle Ser Arg Leu Leu Lys His Asn Pro 355 360 365 Ser Gln Arg Pro Met LeuArg Glu Val Leu Glu His Pro Trp Ile Thr 370 375 380 Ala Asn Ser Ser LysPro Ser Asn Cys Gln Asn Lys Glu Ser Ala Ser 385 390 395 400 Lys Gln Ser11 amino acids amino acid single linear Peptide 5 Glu Asn Ser Tyr ProTrp Pro Tyr Gly Arg Gln 1 5 10 5 amino acids amino acid single linearPeptide 6 Cys Ile Ser Gly Pro 1 5 4 amino acids amino acid single linearPeptide 7 Gln Phe Pro Gln 1 5 amino acids amino acid single linearPeptide 8 Val Asn Ser Gly Gln 1 5 11 amino acids amino acid singlelinear Peptide 9 Arg Lys Glu Pro Val Thr Pro Ser Ala Leu Val 1 5 10 13amino acids amino acid single linear Peptide 10 Leu Met Ser Arg Ser AsnVal Gln Pro Thr Ala Ala Pro 1 5 10 16 amino acids amino acid singlelinear Peptide 11 Val Gln Asn Gln Lys Gln Lys Gln Leu Gln Ala Thr SerVal Pro His 1 5 10 15 11 amino acids amino acid single linear Peptide 12Pro Val Ser Arg Pro Leu Asn Asn Thr Gln Lys 1 5 10 10 amino acids aminoacid single linear Peptide 13 Val Met Glu Asn Ser Ser Gly Thr Pro Asp 15 10 9 amino acids amino acid single linear Peptide 14 Ile Leu Thr ArgHis Phe Thr Ile Asp 1 5 22 amino acids amino acid single linear Peptide15 Ser Lys Gln Pro Leu Pro Ser Ala Pro Glu Asn Asn Pro Glu Glu Gln 1 510 15 Leu Ala Ser Lys Gln Lys 20 23 base pairs nucleic acid singlelinear The letter “R” stands for A or G. The letter “Y” stands for C orT. The letter “N” stands for A, C, G or T. 16 GARTTYGGNG ARGTNTTYYT NGC23 23 base pairs nucleic acid single linear The letter “N” stands for A,C, G or T. The letter “R” stands for A or G. 17 AGNACNCCRA ANGCCCACACRTC 23 8 amino acids amino acid single linear Peptide 18 Glu Phe Gly GluVal Phe Leu Ala 1 5 25 base pairs nucleic acid single linear 19TTTGGCTCGG GAGAAGAAAA GCCAT 25 24 base pairs nucleic acid single linear20 CAATCATCTC TGGGGGCAGG TAGT 24 10 amino acids amino acid single linearPeptide 21 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser 1 5 10 14 amino acidsamino acid single linear Peptide 22 Ser Ala Pro Glu Asn Asn Pro Glu GluGln Leu Ala Ser Lys 1 5 10 13 amino acids amino acid single linearPeptide 23 Arg Pro Leu Asn Asn Thr Gln Lys Ser Lys Gln Pro Leu 1 5 10 13amino acids amino acid single linear Peptide 24 Met Ala Gln Lys Glu AsnSer Tyr Pro Trp Pro Tyr Gly 1 5 10 13 amino acids amino acid singlelinear Peptide 25 Pro Gly Gln Lys Val Met Glu Asn Ser Ser Gly Thr Pro 15 10 8 amino acids amino acid single linear Peptide “Xaa” in positions2, 4, 5 and 7 stands for an unidentified amino acid. 26 Gly Xaa Gly XaaXaa Gly Xaa Val 1 5 9 amino acids amino acid single linear Peptide 27Asp Val Trp Ser Tyr Phe Gly Ile Val 1 5 9 amino acids amino acid singlelinear Peptide “Xaa” in positions 2 and 6 stands for an unidentifiedamino acid. 28 Asp Xaa Trp Ala Ser Xaa Gly Ile Val 1 5 8 amino acidsamino acid single linear Peptide “Xaa” in position 4 represents eitherAsp or Ser. 29 Asp Val Trp Xaa Phe Gly Val Leu 1 5 21 base pairs nucleicacid single linear 30 CAGGGCAGAG TGGTCACTTT C 21 21 base pairs nucleicacid single linear 31 CGTCCGCCAC TCCGACCAGC C 21 21 base pairs nucleicacid single linear 32 CTGCAGTCGAA CCTTGCCTCC A 21 8 amino acids aminoacid single linear Peptide 33 Asp Val Trp Ala Phe Gly Val Leu 1 20 basepairs nucleic acid single linear 34 CGCCTTTGCA TCCGCTCCTG 20 20 basepairs nucleic acid single linear 35 GATTTGCCTC CTGTGAAGAC 20 21 basepairs nucleic acid single linear 36 ATGCCTCCGG AAAGAGCCTG T 21 22 basepairs nucleic acid single linear 37 GTGTCCCACT GCTATTCTCC AT 22 21 basepairs nucleic acid single linear 38 CAGGGCTGCC ATATAACCTG A 21 20 basepairs nucleic acid single linear 39 CTAGCACAGG CTGACGGGGC 20

What is claimed is:
 1. An isolated, enriched or purified nucleic acidmolecule encoding AUR1 and/or AUR2 polypeptide.
 2. The nucleic acidmolecule of claim 1, wherein said nucleic acid molecule comprises anucleotide sequence that: (a) encodes a polypeptide having the fulllength amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4; (b)is the complement of the nucleotide sequence of (a); (c) hybridizesunder highly stringent conditions to the nucleotide molecule of (a) andencodes a naturally occurring AUR1 and/or AUR2 polypeptide; (d) encodesAUR1 and/or AUR2 polypeptide having the full length amino acid sequenceof SEQ ID NO:3 or SEQ ID NO:4 except that it lacks one or more of thefollowing segments of amino acid residues: 1-73, 74-271, or 272-344 ofSEQ ID NO:3, or 1-129, 130-274, or 275-403 of SEQ ID 20 NO:4; (e) is thecomplement of the nucleotide sequence of (d); (f) encodes a polypeptidehaving the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4from amino acid residues 1-73, 74-271, or 272-344 of SEQ ID NO:3, or1-129, 130-274, or 275-403 of SEQ ID NO:4; (g) is the complement of thenucleotide sequence of (f); (h) encodes a polypeptide having the fulllength amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4except that it lacks one or more of the domains selected from the groupconsisting of a C-terminal domain, a catalytic domain, and an N-terminaldomain; or (i) is the complement of the nucleotide sequence of (h). 3.The nucleic acid molecule of claim 1, further comprising a vector orpromoter effective to initiate transcription in a host cell.
 4. Thenucleic acid molecule of claim 1 or claim 2, wherein said nucleic acidmolecule is isolated, enriched, or purified from a mammal.
 5. Thenucleic acid molecule of claim 4, wherein said nucleic acid molecule isisolated, enriched, or purified from a human.
 6. A nucleic acid probefor the detection of nucleic acid encoding AUR1 and/or AUR2 polypeptidein a sample.
 7. The probe of claim 6, wherein said polypeptide is afragment of the protein encoded by the full length amino acid sequenceset forth in SEQ ID NO:3 or SEQ ID NO:4.
 8. A recombinant cellcomprising a nucleic acid molecule encoding AUR1 and/or AUR2polypeptide.
 9. The cell of claim 8, wherein said polypeptide is afragment of the protein encoded by the full length amino acid sequenceset forth in SEQ ID NO:3 or SEQ ID NO:4.
 10. An isolated, enriched, orpurified AUR1 or AUR2 polypeptide.
 11. The polypeptide of claim 10,wherein said polypeptide is a fragment of the protein encoded by thefull length amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4.12. The polypeptide of claim 10, wherein said polypeptide comprises anamino acid sequence having (a) the full length amino acid sequence setforth in SEQ ID NO:3 or SEQ ID NO:4; (b) the full length amino acidsequence set forth in SEQ ID NO:3 or SEQ ID NO:4 except that it lacksone or more of the following segments of amino acid residues: 1-73,74-271, or 272-344 of SEQ ID NO:3, or 1-129, 130-274, or 275-403 of SEQID NO:4; (c) the amino acid sequence set forth in SEQ ID NO:3 or SEQ IDNO:4 from amino acid residues 1-73, 74-271, or 272-344 of SEQ ID NO:3,or 1-129, 130-274, or 275-403 of SEQ ID NO:4; or (d) the full lengthamino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4 except thatit lacks one or more of the domains selected from the group consistingof a C-terminal domain, a catalytic domain, and an N-terminal domain.13. The AUR1 or AUR2 polypeptide of claim 10, wherein said polypeptideis isolated, purified, or enriched from a mammal.
 14. The AUR1 or AUR2polypeptide of claim 13, wherein said polypeptide is isolated, purified,or enriched from a human.
 15. The AUR1 or AUR2 polypeptide of claim 10,wherein said polypeptide is an AUR1 polypeptide.
 16. The AUR1 or AUR2polypeptide of claim 10, wherein said polypeptide is an AUR2polypeptide.
 17. An antibody or antibody fragment having specificbinding affinity to AUR1 and/or AUR2 polypeptide or AUR1 and/or AUR2domain polypeptide.
 18. A hybridoma which produces an antibody havingspecific binding affinity to an AUR1 and/or AUR2 polypeptide.
 19. Amethod for identifying a substance that modulates AUR1 and/or AUR2activity comprising the steps of: (a) contacting AUR1 and/or AUR2polypeptide with a test substance; (b) measuring the activity of saidpolypeptide; and (c) determining whether said substance modulates theactivity of said polypeptide.
 20. A method for identifying a substancethat modulates AUR1 and/or AUR2 activity in a cell comprising the stepsof: (a) expressing AUR1 and/or AUR2 polypeptide in a cell; (b) adding atest substance to said cell; and (c) monitoring a change in cellphenotype or the interaction between AUR1 and/or AUR2 polypeptide and anatural binding partner.
 21. A method of treating disease byadministering to a patient in need of such treatment a substance thatmodulates the activity of AUR1 and/or AUR2.
 22. The method of claim 21,wherein said disease is selected from the group consisting of colon,breast, renal, ovarian, bladder, head and neck cancers, and gliomas,medulloblastomas, chondrosarcomas, and pancreatic tumors.
 23. The methodof claim 21, wherein said disease is selected from the group consistingof colon, breast and renal cancer.
 24. The method of claim 21, whereinsaid substance is an antisense oligonucleotide selected from the groupconsisting of: SEQ ID NO:30, SEQ ID NO: 31, and SEQ ID NO:32.
 25. Themethod of claim 21, wherein said substance is a protein kinaseinhibitor.
 26. A method for detection of AUR1 and/or AUR2 in a sample asa diagnostic tool for diseases comprising the steps of: (a) contactingsaid sample with a nucleic acid probe which hybridizes underhybridization assay conditions to a nucleic acid target region of aur1and/or aur2, said probe comprising the nucleic acid sequence encodingAUR1 and/or AUR2 polypeptide, fragments thereof, and the complements ofsaid sequences and fragments; and (b) detecting the presence or amountof the probe:target region hybrid as an indication of said disease. 27.The method of claim 25, wherein said disease is colon cancer.
 28. Anantisense oligonucleotide comprised of a nucleotide base sequenceselected from the group consisting of SEQ ID NO:30, SEQ ID NO:31, andSEQ ID NO:32.